VMP-like sequences of pathogenic borrelia

ABSTRACT

The present invention relates to DNA sequences encoding Vmp-like polypeptides of pathogenic Borrelia, the use of the DNA sequences in recombinant vectors to express polypeptides, the encoded amino acid sequences, application of the DNA and amino acid sequences to the production of polypeptides as antigens for immunoprophylaxis, immunotherapy, and immunodiagnosis. Also disclosed are the use of the nucleic acid sequences as probes or primers for the deletion of organisms causing Lyme disease, relapsing fever, or related disorders, and kits designed to facilitate methods of using the described polypeptides, DNA segments and antibodies.

This application claims the benefit of provisional application60/012,028 filed Feb. 21, 1996.

The government owns rights in the present invention pursuant to grantnumber NIH2R01 AI37277-04 from the National Institutes of health.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to the field of molecular biology; in particular,to immunogenic compositions and recombinant VMP-like genes useful fortreatment and diagnosis of Lyme disease. Also included are methods forthe determination of virulence factors in Lyme disease.

2. Description of Related Art

Lyme disease is a bacterial infection caused by pathogenic spirochetesof the genus Borrelia. The infection can occur in humans, dogs, deer,mice and other animals, and is transmitted by arthropod vectors, mostnotably ticks of the genus Ixodes. Borrelia burgdorferi, the most commoncause of Lyme disease in North America, was first cultured in 1982. B.garinii and B. afzelii are the most common infectious agents of Lymedisease in Europe, and another species, B. japonicum, has been describedin Japan. These organisms are closely related and cause similarmanifestations with multiple stages: an expanding rash at the site ofthe tick bite (erythema migrans); fever, lymphadenopathy, fatigue, andmalaise; effects of disseminated infection, including carditis,meningoradiculitis, and polyarthritis; and chronic manifestationsincluding arthritis and neurologic disorders. Lyme disease is oftendifficult to diagnose because of shared manifestations with otherdisorders, and it can also be refractory to treatment during late stagesof the disease. It is most common in areas such as suburban regions ofupstate New York and Connecticut, where large populations of deer andwhite-footed mice serve as the principal mammalian hosts and reservoirsof infection. Approximately 10,000 cases of Lyme disease in humans arereported per year in the United States. and it is also a significantveterinary problem due to a high infection rate of dogs and otherdomestic animals in endemic regions.

B. burgdorferi, the etiologic agent of Lyme disease, is able to persistfor years in patients or animals despite the presence of an activeimmune response (Steer, 1989; Schutzer, 1992). Antigenic variation hasbeen postulated previously as a mechanism whereby B. burgdorferi evadesthe immune response in the mammalian host (Schwan et al., 1991; Wilskeet al., 1992). Antigenic variation has been defined as changes in thestructure or expression of antigenic proteins that occurs duringinfection at a frequency greater than the usual mutation rate (Borst andGeaves, 1987; Robertson and Meyer, 1992; Seifert and So, 1988).

Relapsing fever is another disease caused by pathogenic Borrelia. It hasboth epidemic and endemic forms. The epidemic form is caused by B.recurrentis and is transmitted between humans by lice. It was a majorsource of morbidity and mortality during World War I, but has been raresince then due largely to public health measures. Endemic relapsingfever is an epizootic infection caused by several Borreliae species,including B. hermsii. It occurs sporadically among hunters, spelunkers,and others who come in contact with infected soft-bodied ticks of thegenus Ornithidorus. Relapsing fever is characterized by two or moreepisodes or “relapses” of high bacteremia (up to 10⁸/ml). The first waveof infection is caused by Borreliae expressing a certain Variable MajorProtein (VMP) on their surface (e.g. Vmp21). The gene encoding this VMPis located at a promoter site in the expression plasmid, whereas over 24nonexpressed copies of different VMP genes are present on the so-calledsilent plasmid. When the host develops antibodies against the expressedVMP, the organisms of that stereotype are destroyed and the patientimproves. However, a small proportion of organisms have undergoneantigenic switching to a different stereotype. Nonreciprocalrecombination occurs between the expression plasmid and the silentplasmid, resulting in the insertion of a different VMP gene in theexpression site (e.g., Vmp7). The organisms expressing Vmp7 are notaffected by the anti-Vmp21 antibodies, and therefore multiply in thehost and cause a second episode of the disease. Up to five of these 3-5day episodes can occur, separated by 1-2 week intervals.

Such well-demarcated episodes of infection do not occur during Lymedisease, and fewer organisms are present in the blood and in tissues atany stage. However, there are reasons to suspect that similar mechanismsof antigenic variation may occur in B. burgdorferi and other Lymedisease Borreliae. The infection, if untreated, commonly persists formonths to years despite the occurrence of host antibody and cellularresponses; this observation indicates effective evasion of the immuneresponse. Lyme disease may be disabling (particularly in its chronicform), and thus there is a need for effective therapeutic andprophylactic treatment.

Certain B burgdorferi genes and proteins have been patented, includingOuter Surface Protein D (OspD) (U.S. Pat. No. 5,246,844; issued Sep. 21,1993). OspD has not proven to be a useful protein for diagnosis orimmunoprotection. Other proteins, including OspA and OspC, have beenconsidered as vaccine candidates for Lyme disease, including arecombinant OspA vaccine currently in human clinical trials. Othervaccines are in use or undergoing testing in veterinary applications,including vaccination of dogs. However, animal studies indicate thatOspA vaccination may not be effective against all strains of Lymedisease Borreliae. OspA is also not useful for immunodiagnosis, due toweak antibody responses to OspA in Lyme disease patients.

Previous studies have generally failed to provide evidence for theoccurrence of antigenic variation in Lyme disease Borrelia. Geneticheterogeneity in the genes encoding the membrane lipoproteins OspA,OspB, OspC, and OspD has been well documented among strains of Lymedisease Borreliae (Marconi et al., 1993; Marconi et al., 1994; Livey etal., 1995). In addition, mutations in ospA and ospB have been shown tooccur in vitro (Rosa et al., 1992; Sadziene et al., 1992). However, nosignificant antigenic change (Barthold, 1993) or gross geneticalteration (Persing et al., 1994; Stevenson et al., 1994) has beendetected in B. burgdorferi N40 isolates from chronically infected BALB/cand C3H mice, other than the loss of the 38-kilobase (kb) plasmidencoding OspD. Therefore the heterogeneity in Osp proteins observedamong B. burgdorferi sensu lato isolates appears to representevolutionary divergence (“antigenic drift”) rather than antigenicvariation.

There is a commercial demand for vaccines and diagnostic kits for Lymedisease, both for human and veterinary use. Several companies haveactive research and development programs in these areas.

SUMMARY OF THE INVENTION

Partial and complete DNA sequences have been determined for severalrecombinant clones containing DNA encoding VMP-like sequences. Theidentification and characterization of these sequences now allows: (1)identification of the expressed gene(s) in B. burgdorferi; (2)expression of these gene(s) by a recombinant vector in a host organismsuch as E. coli; (3) immunization of laboratory animals with theresulting polypeptide, and determination of protective activity againstB. burgdorferi infection; (4) use of antibodies against the expressedprotein to identify the reactive polypeptide(s) in B. burgdorferi cells;(5) use of the expressed protein(s) to detect antibody responses ininfected humans and animals; (6) determination of the presence, sequencedifferences, and expression of the VMP-like DNA sequences in other Lymedisease Borreliae.

The invention is contemplated to be useful in the immunoprophylaxis,diagnosis, or of Lyme disease, relapsing fever, or related diseases inhumans or animals. It is expected that recombinant or native proteinsexpressed by the VMP-like genes (or portions thereof) will be useful for(a) immunoprophylaxis against Lyme disease, relapsing fever, or relateddisorders in humans and animals; (b) immunotherapy of existing Lymedisease, relapsing fever, or related illnesses, by way of immunizationof injection of antibodies directed against VMP-like proteins; and (c)immunodiagnosis of Lyme disease, relapsing fever, or related diseases,including their use in kits in which the VMP-like proteins are the soleantigen or one of multiple antigens. The DNA may be employed in: (a)production of recombinant DNA plasmids or other vectors capable ofexpressing recombinant polypeptides; and (b) design and implementationof nucleic acid probes or oligonucleotides for detection and/oramplification of VMP-like sequences. The latter is expected to haveapplication in the diagnosis of infection with Borrelial organisms.

Similar sequences in B. burgdorferi and other Lyme disease Borreliaehave not been reported previously, as determined by BLAST searches ofcurrent nucleotide and amino acid databases including Genbank, the EMBLDNA database, and the Swiss Protein database. Although there is somesimilarity between the B. burgdorferi deduced amino acid sequences withpreviously published B. hermsii VMP deduced amino acid sequences, thedegree of identity and similarity is only ˜30% and ˜50%, respectively.Outer surface protein C (OspC) of Lyme disease organisms has beenreported to have sequence similarities to VMPs, but the highestsimilarity is to a different subgroup of VMPs than the sequencesreported here (Carter et al., 1994). The VMP-like sequences such asthose contained in pJRZ53-31 have a low degree of homology with OspCfrom some Lyme disease organisms (e.g. B. burgdorferi 2591), asindicated by a BLASTP homology score of 60 and a probability of 0.0013.Thus, the B. burgdorferi VMP-like DNA sequences are unique, althoughthey have an apparent evolutionary relationship with other Borreliagenes.

Another aspect of the invention is the method for identification ofpossible virulence factors. This approach entails subtractivehybridization of target DNA from high infectivity organisms with driverDNA from low-infectivity strains or clones. This procedure greatlyenriches for sequences which differ between the high- andlow-infectivity strains and thus may encode proteins important invirulence. Of particular utility is the use of closely related isogenicclones that differ in their infectivity; in this case, the DNAdifferences should be restricted more stringently to those related toinfectivity.

Open reading frames in a B. burgdorferi plasmid that encode hypotheticalproteins resembling the VMP proteins of relapsing fever organisms havenow been identified. The inventors have found that the presence of theplasmid containing these VMP-like sequences in B. burgdorferi clonescorrelates strongly with infectivity. Thus it is likely that theproteins encoded by the VMP-like sequences are important inimmunoprotection and pathogenesis. Proteins encoded by the VMP-likesequences of B. burgdorferi may provide protection when used eitheralone or in combination with other antigens. They may also be useful forimmunodiagnosis.

The invention is considered to include DNA segments corresponding to 20,30, and 40 base pairs of the VMP-like sequences; DNA segments inclusiveof the entire open leading frames of the VMP-like sequences; amino acidsequences corresponding to both conserved and variable regions of theVMP-like sequences; recombinant vectors encoding an antigenic proteincorresponding to the above amino acid sequences; recombinant cells whereextrachromosomal DNA expresses a polypeptide encoded by the DNA encodingBorrelia VMP-like sequences; a recombinant B. burgdorferi or E. colicell containing the DNA encoding VMP-like sequences; methods ofpreparing transformed bacterial host cells using the DNA encoding theVMP-like polypeptides; methods using the plasmid or vector to transformthe bacterial host cell to express B. burgdorferi polypeptides encodedby the DNA sequences; methods for immunization of humans or animals withthe native B. burgdorferi polypeptide or polypeptides expressed byrecombinant cells that include DNA encoding the VMP-like polypeptides;and methods for identifying potential virulence factors usingsubtractive hybridization between target DNA from high-infectivity cellsand driver DNA from low-infectivity cells.

Also included in the invention are primer sets capable of primingamplification of the VMP-like DNA sequences; kits for the detection ofB. burgdorferi nucleic acids in a sample, the kits containing a nucleicacid probe specific for the VMP-like sequences, together with a meansfor detecting a specific hybridization with the probe; kits fordetection of antibodies against the VMP-like sequences of B. burgdorferiand kits containing a native or recombinant VMP-like polypeptide,together with means for detecting a specific binding of antibodies tothe antigen.

Methods of Treatment

An important aspect of the invention is the recognition that BorreliaVMP-like sequences recombine at the vls site, with the result thatantigenic variation is virtually limitless. Multiclonal populationstherefore can exist in an infected patient so that immunologicaldefenses are severely tested if not totally overwhelmed. Thus there isnow the opportunity to develop more effective combinations of immunogensfor protection against Borrelia infections or as preventive inoculationssuch as in the form of cocktails of multiple antigenic variants based ona base series of combinatorial VMP-like antigens.

VMP-like protein preparations may be administered in several ways,either locally or systematically in pharmaceutically acceptableformulations. Amounts appropriate for administration are determined onan individual basis depending on such factors as age and sex of thesubject, as well as physical condition and weight. Such determinationsare well within the skill of the practitioner in the medical field.

Other methods of administration may include injection of BorreliaVMP-like DNAs into vaccine recipients (human or animal) driven by anappropriate promoter such as CMV, (so called DNA vaccines). Suchpreparations could be injected directly into lesions or transplantedinto patients for systemic immunization. DNA vaccinations techniques arecurrently well past the initial development stage and have shown promiseas vaccination strategies.

VMP-like Genes

Recombinant proteins and polypeptides encoded by isolated DNA segmentsand genes are often referred to with the prefix “r” for recombinant. Assuch, DNA segments encoding rVMPs, or rVMP-related genes, etc. arecontemplated to be particularly useful in connection with thisinvention. Any recombinant vls combining any of the vlsE expression siteloci and/or silent vls cassettes (vls2-vls-16) gene would likewise bevery useful with the methods of the invention.

Isolation of the DNA encoding VMP polypeptides allows one to use methodswell known to those of skill in the art and as herein described to makechanges in the codons for specific amino acids such that the codons are“preferred usage” codons for a given species. Thus for example,preferred codons will vary significantly for bacterial species ascompared with mammalian species however, there are preferences evenamong related species. Shown below is a preferred codon usage tablehuman. Isolation of spirochete DNA encoding VMP will allow substitutionsfor preferred human codons, although expressed polypeptide product fromhuman DNA is expected to be homologous to bacterial VMP and so would beexpected to be structurally and functionally equivalent to VMP isolatedfrom a spirochete. However, substitutions of preferred human codons mayimprove expression in the human host, thereby improving the efficiencyof potential DNA vaccines.

TABLE 1 Homo sapiens Codon υ^(b) Total #^(a) Codon υ^(b) Total #^(a)Codon υ^(a) Total #^(a) Codon υ^(b) Total #^(a) UUU 16.6 72711 UCU 14.062953 UAU 12.3 55039 UGU  9.5 42692 UUC 21.4 95962 UCC 17.7 79482 UAC17.0 76480 UGC 12.8 57368 UUA  6.3 28202 UCA 10.7 48225 UAA  0.7  2955UGA  1.2  5481 UUG 11.5 51496 UCG  4.4 19640 UAG  0.5  2181 UGG 13.559982 CUU 11.7 52401 CCU 16.7 74975 CAU  9.6 43193 CGU  4.6 20792 CUC19.5 87696 CCC 20.0 89974 CAC 14.6 65533 CGC 11.0 49507 CUA  6.3 28474CCA 16.2 72711 CAA 11.4 51146 CGA  5.9 26551 CUG 40.6 182139  CCG  6.930863 CAG 33.7 151070  CGG 11.3 50682 AUU 15.7 70652 ACU 12.8 57288 AAU16.6 74401 AGU 11.1 49894 AUC 23.7 106390  ACC 21.1 94793 AAC 21.1 94725AGC 19.1 85754 AUA  6.7 30139 ACA 14.7 66136 AAA 23.2 104221  AGA 10.848369 AUG 22.6 101326  ACG  6.7 30059 AAG 33.9 152179  AGG 10.9 48882GUU 10.6 47805 GCU 18.7 83800 GAU 22.0 98712 GCU 11.2 50125 GUC 15.670189 GCC 29.2 130966  GAC 27.0 121005  GGC 24.0 107571  GUA  6.6 29659GCA 15.3 68653 GAA 27.8 124852  GGA 16.9 75708 GUG 30.0 134750  GCG  7.533759 GAG 40.8 182943  GGG 16.7 74859 Coding GC 52.96% 1st letter GC55.98% 2nd letter GC 42.29% 3rd letter GC 60.60% ^(a)Total 4489120 ^(b)υ= Frequency per 1000

The definition of a “VMP-like gene”, “VMP-related gene” as used herein,is a gene that hybridizes, under relatively stringent hybridizationconditions (see, e.g., Maniatis et al., 1982), to DNA sequencespresently known to include related gene sequences.

To prepare an VMP-like gene segment or cDNA one may follow the teachingsdisclosed herein and also the teachings of any of patents or scientificdocuments specifically referenced herein. One may obtain a rVMP- orother related-encoding DNA segments using molecular biologicaltechniques, such as polymerase chain reaction (PCR™) or screening of acDNA or genomic library, using primers or probes with sequences based onthe above nucleotide sequence. Such fragments may be readily preparedby, for example, directly synthesizing the fragment by chemical means,by application of nucleic acid reproduction technology, such as the PCR™technology of U.S. Pat. Nos. 4,683,195 and 4,683,202 (hereinincorporated by reference). The practice of these techniques is aroutine matter for those of skill in the art, as taught in variousscientific texts (see e.g., Sambrook et al., 1989), incorporated hereinby reference. Certain documents further particularly describe suitablemammalian expression vectors, e.g., U.S. Pat. No. 5,168,050,incorporated herein by reference. The VMP genes and DNA segments thatare particularly preferred for use in certain aspects of the presentmethods are those encoding VMP and VMP-related polypeptides.

It is also contemplated that one may clone other additional genes orcDNAs that encode a VMP or VMP-related peptide, protein or polypeptide.The techniques for cloning DNA molecules, i.e., obtaining a specificcoding sequence from a DNA library that is distinct from other portionsof DNA, are well known in the art. This can be achieved by, for example,screening an appropriate DNA library which relates to the cloning of avls gene such as from the variable region of that gene. The screeningprocedure may be based on the hybridization of oligonucleotide probes,designed from a consideration of portions of the amino acid sequence ofknown DNA sequences encoding related Borrelia proteins. The operation ofsuch screening protocols is well known to those of skill in the art andare described in detail in the scientific literature, for example, seeSambrook et al., 1989.

Techniques for introducing changes in nucleotide sequences that aredesigned to alter the functional properties of the encoded proteins orpolypeptides are well known in the art, e.g., U.S. Pat. No. 4,518,584,incorporated herein by reference, which techniques are also described infurther detail herein. Such modifications include the deletion,insertion or substitution of bases, and thus, changes in the amino acidsequence. Changes may be made to increase the VMP activity of a protein,to increase its biological stability or half-life, to change itsglycosylation pattern, and the like. All such modifications to thenucleotide sequences are encompassed by this invention.

VMP-encoding DNA Segments

The present invention, in a general and overall sense, also concerns theisolation and characterization of novel vls gene segments, which encodecombinatorial mosaics of expressed and silent regions of the vls gene. Apreferred embodiment of the present invention is a purified nucleic acidsegment that encodes a protein that has at least a partial amino acidsequence in accordance with SEQ ID NO:2. Another embodiment of thepresent invention is a purified nucleic acid segment, further defined asincluding nucleotide sequences in accordance with SEQ ID NO:1 and SEQ IDNO:3.

In a more preferred embodiment the purified nucleic acid segmentconsists essentially of the nucleotide sequence of SEQ ID NO:1 and SEQID NO:3, their complement or the degenerate variants thereof. As usedherein, the term “nucleic acid segment” and “DNA segment” are usedinterchangeably and refer to a DNA molecule which has been isolated freeof total genomic DNA of a particular species. Therefore, a “purified”DNA or nucleic acid segment as used herein, refers to a DNA segmentwhich contains a VMP coding sequence yet is isolated away from, orpurified free from, total genomic DNA, for example, total cDNA orborrelia genomic DNA. Included within the term “DNA segment”, are DNAsegments and smaller fragments of such segments, and also recombinantvectors, including, for example, plasmids, cosmids, phage, viruses, andthe like.

Similarly, a DNA segment comprising an isolated or purified vls generefers to a DNA segment including VMP-related coding sequences isolatedsubstantially away from other naturally occurring genes or proteinencoding sequences. In this respect, the term “gene” is used forsimplicity to refer to a functional protein, polypeptide or peptideencoding unit. As will be understood by those in the art, thisfunctional term includes both genomic sequences, cDNA sequences orcombinations thereof. “Isolated substantially away from other codingsequences” means that the gene of interest, in this case vls, forms thesignificant part of the coding region of the DNA segment, and that theDNA segment does not contain large portions of naturally-occurringcoding DNA, such as large chromosomal fragments or other functionalgenes or cDNA coding regions. Of course, this refers to the DNA segmentas originally isolated, and does not exclude genes or coding regionslater added to the segment by the hand of man, nor are other portions orcontiguous sequences of naturally occurring DNA excluded.

In particular embodiments, the invention concerns isolated DNA segmentsand recombinant vectors incorporating DNA sequences which encode aVMP-like protein that includes within its amino acid sequence an aminoacid sequence in accordance with SEQ ID NO:2.

Another preferred embodiment of the present invention is a purifiednucleic acid segment that encodes a protein in accordance with SEQ IDNO:2, further defined as a recombinant vector. As used herein the term,“recombinant vector”, refers to a vector that has been modified tocontain a nucleic acid segment that encodes an VMP protein, or afragment thereof. The recombinant vector may be further defined as anexpression vector comprising a promoter operatively linked to saidVMP-encoding nucleic acid segment.

A further preferred embodiment of the present invention is a host cell,made recombinant with a recombinant vector comprising an vls gene. Therecombinant host cell may be a prokaryotic cell. As used herein, theterm “engineered” or “recombinant” cell is intended to refer to a cellinto which a recombinant gene, such as a gene encoding VMP, has beenintroduced. Therefore, engineered cells are distinguishable fromnaturally occuring cells which do not contain a recombinantly introducedgene. Engineered cells are thus cells having a gene or genes introducedthrough the hand of man. Recombinantly introduced genes will either bein the form of a copy of a genomic gene or a cDNA gene, or will includegenes positioned adjacent to a promoter not naturally associated withthe particular introduced gene.

In certain embodiments, the invention concerns isolated DNA segments andrecombinant vectors which encode a protein or peptide that includeswithin its amino acid sequence an amino acid sequence essentially as setforth in SEQ ID NO:2. Naturally, where the DNA segment or vector encodesa full length VMP-like protein, or is intended for use in expressing theVMP-like protein, the most preferred sequences are those which areessentially as set forth in SEQ ID NO:2. It is recognized that SEQ IDNO:2 represents the full length protein encoded by the vls gene and thatcontemplated embodiments include up to the full length sequence andfunctional variants as well.

The term “a sequence essentially as set forth in SEQ ID NO:2” means thatthe sequence substantially corresponds to a portion of SEQ ID NO:2 andhas relatively few amino acids which are not identical to, or abiologically functional equivalent of, the amino acids of SEQ ID NO:2.The term “biologically functional equivalent” is well understood in theart and is further defined in detail herein, as a gene having a sequenceessentially as set forth in SEQ ID NO:1 and that is associated with avls gene in the Borrelia family. Accordingly, sequences which havebetween about 70% and about 80%; or more preferably, between about 85%and about 90%; or even more preferably, between about 90 and 95% andabout 99%; of amino acids which are identical or functionally equivalentto the amino acids of SEQ ID NO:2 will be sequences which are“essentially as set forth in SEQ ID NO:2”.

In certain other embodiments, the invention concerns isolated DNAsegments and recombinant vectors that include within their sequence anucleic acid sequence essentially as set forth in SEQ ID NO:1 and SEQ IDNO:3. The term “essentially as set forth in SEQ ID NO:1 and SEQ ID NO3,”is used in the same sense as described above and means that the nucleicacid sequence substantially corresponds to a portion of SEQ ID NO:1 andSEQ ID NO:3, and has relatively few codons which are not identical, orfunctionally equivalent, to the codons of SEQ ID NO:1 and SEQ ID NO:3.The term “functionally equivalent codon” is used herein to refer tocodons that encode the same amino acid, such as the six codons forarginine or serine, as set forth in Table 4, and also refers to codonsthat encode biologically equivalent amino acids.

It will also be understood that amino acid and nucleic acid sequencesmay include additional residues, such as additional N- or C-terminalamino acids or 5′ or 3′ sequences, and yet still be essentially as setforth in one of the sequences disclosed herein, so long as the sequencemeets the criteria set forth above, including the maintenance ofbiological protein activity where protein expression is concerned. Theaddition of terminal sequences particularly applies to nucleic acidsequences which may, for example, include various non-coding sequencesflanking either of the 5′ or 3′ portions of the coding region or mayinclude various internal sequences, i.e., introns, which are known tooccur within genes.

Excepting intronic or flanking regions, and allowing for the degeneracyof the genetic code, sequences which have between about 70% and about80%; or more preferably, between about 80%, 85% and about 90%; or evenmore preferably, between about 90%, 95% and about 99%; of nucleotideswhich are identical to the nucleotides of SEQ ID NO:1 and SEQ ID NO:3will be sequences which are “essentially as set forth in SEQ ID NO:1 andSEQ ID NO:3”. Sequences which are essentially the same as those setforth in SEQ ID NO:1 and SEQ ID NO:3 may also be functionally defined assequences which are capable of hybridizing to a nucleic acid segmentcontaining the complement of SEQ ID NO:1 and SEQ ID NO:3 underrelatively stringent conditions or conditions of high stringency.Suitable relatively stringent hybridization conditions will be wellknown to those of skill in the art and are clearly set forth herein, forexample conditions for use with Southern and Northern blot analysis, andas described in the examples herein set forth.

Naturally, the present invention also encompasses DNA segments which arecomplementary, or essentially complementary, to the sequence set forthin SEQ ID NO:1 and SEQ ID NO:3. Nucleic acid sequences which are“complementary” are those which are capable of base-pairing according tothe standard Watson-Crick complementary rules. As used herein the term“complementary sequences” means nucleic acid sequences which aresubstantially complementary, as may be assessed by the same nucleotidecomparison set forth above, or as defined as being capable ofhybridizing to the nucleic acid segment of SEQ ID NO:1 and SEQ ID NO:3under relatively stringent conditions, i.e., conditions of highstringency.

The nucleic acid segments of the present invention, regardless of thelength of the coding sequence itself, may be combined with other DNAsequences, such as promoters, polyadenylation signals, additionalrestriction enzyme sites, multiple cloning sites, other coding segments,and the like, such that their overall length may vary considerably. Itis therefore contemplated that a nucleic acid fragment of almost anylength may be employed, with the total length preferably being limitedby the ease of preparation and use in the intended recombinant DNAprotocol. For example, nucleic acid fragments may be prepared whichinclude a short stretch complementary to SEQ ID NO:1 and SEQ ID NO:3,such as about 10 to 15 or 20, 30, or 40 or so nucleotides, and which areup to 2000 or so base pairs in length. DNA segments with total lengthsof about 8000, 7000, 6000, 5000, 4000, 3000, 2000, 1000, 500, 200, 100and about 50 base pairs in length are also contemplated to be useful.

A preferred embodiment of the present invention is a nucleic acidsegment which comprises at least a 14-nucleotide long stretch whichcorresponds to, or is complementary to, the nucleic acid sequence of SEQID NO:1 and SEQ ID NO:3. In a more preferred embodiment the nucleic acidis further defined as comprising at least a 20 nucleotide long stretch,a 30 nucleotide long stretch, 50 nucleotide long stretch, 100 nucleotidelong stretch, or at least an 2000 nucleotide long stretch whichcorresponds to, or is complementary to, the nucleic acid sequence of SEQID NO:1 and SEQ ID NO:3. The nucleic acid segment may be further definedas having the nucleic acid sequence of SEQ ID NO:1 and SEQ ID NO:3.

A related embodiment of the present invention is a nucleic acid segmentwhich comprises at least a 14-nucleotide long stretch which correspondsto, or is complementary to, the nucleic acid sequence of SEQ ID NO:1 andSEQ ID NO:3, further defined as comprising a nucleic acid fragment of upto 10,000 basepairs in length. A more preferred embodiment if a nucleicacid fragment comprising from 14 nucleotides of SEQ ID NO:1 and SEQ IDNO:3 up to 5,000 basepairs in length, 3,000 basepairs in length, 2,000basepairs in length, 1,000 basepairs in length, 500 basepairs in length,or 100 basepairs in length.

Naturally, it will also be understood that this invention is not limitedto the particular nucleic acid and amino acid sequences of SEQ ID NO:1and SEQ ID NO:3. Recombinant vectors and isolated DNA segments maytherefore variously include the VMP-like protein coding regionsthemselves, coding regions bearing selected alterations or modificationsin the basic coding region, or they may encode larger polypeptides whichnevertheless include VMP-coding regions or may encode biologicallyfunctional equivalent proteins or peptides which have variant aminoacids sequences.

The DNA segments of the present invention encompass biologicallyfunctional equivalent VMP-like proteins and peptides. Such sequences mayarise as a consequence of codon redundancy and functional equivalencywhich are known to occur naturally within nucleic acid sequences and theproteins thus encoded. Alternatively, functionally equivalent proteinsor peptides may be created via the application of recombinant DNAtechnology, in which changes in the protein structure may be engineered,based on considerations of the properties of the amino acids beingexchanged. Changes designed by man may be introduced through theapplication of site-directed mutagenesis techniques, e.g., to introduceimprovements to the antigenicity of the VMP-like protein or to testVMP-like mutants in order to examine activity or determine the presenceof VMP-like peptide in various cells and tissues at the molecular level.

A preferred embodiment of the present invention is a purifiedcomposition comprising a polypeptide having an amino acid sequence inaccordance with SEQ ID NO:2. The term “purified” as used herein, isintended to refer to an VMP-related protein composition, wherein theVMP-like protein is purified to any degree relative to itsnaturally-obtainable state, i.e., in this case, relative to its puritywithin a eukaryotic cell extract. A preferred cell for the isolation ofVMP-like protein is from borrelia organisms; however, VMP-like proteinmay also be isolated from various patient specimens, specimens frominfected animals, recombinant cells, tissues, isolated subpopulations oftissues, and the like, as will be known to those of skill in the art, inlight of the present disclosure. A purified VMP-like protein compositiontherefore also refers to a polypeptide having the amino acid sequence ofSEQ ID NO:2, free from the environment in which it may naturally occur.

If desired, one may also prepare fusion proteins and peptides, e.g.,where the VMP-like protein coding regions are aligned within the sameexpression unit with other proteins or peptides having desiredfunctions, such as for purification or immunodetection purposes (e.g.,proteins which may be purified by affinity chromatography and enzymelabel coding regions, respectively).

Turning to the expression of the vls gene whether from cDNA based orgenomic DNA, one may proceed to prepare an expression system for therecombinant preparation of VMP-like protein. The engineering of DNAsegment(s) for expression in a prokaryotic or eukaryotic system may beperformed by techniques generally known to those of skill in recombinantexpression. For example, one may prepare a VMP-GST(glutathione-S-transferase) fusion protein that is a convenient means ofbacterial expression. However, it is believed that virtually anyexpression system may be employed in the expression of VMP-likeproteins.

VMP-like proteins may be successfully expressed in eukaryotic expressionsystems, however, the inventors contemplate that bacterial expressionsystems may be used for the preparation of VMP for all purposes. ThecDNA containing vls gene may be separately expressed in bacterialsystems, with the encoded proteins being expressed as fusions withβ-galactosidase, avidin, ubiquitin, Schistosoma japonicum glutathioneS-transferase, multiple histidines, epitope-tags and the like. It isbelieved that bacterial expression will ultimately have advantages overeukaryotic expression in terms of ease of use and quantity of materialsobtained thereby.

It is proposed that transformation of host cells with DNA segmentsencoding VMP-like proteins will provide a convenient means for obtaininga VMP-like protein. It is also proposed that cDNA, genomic sequences,and combinations thereof, modified by the addition of a eukaryotic orviral promoter, are suitable for eukaryotic expression, as the host cellwill, of course, process the genomic transcripts to yield functionalmRNA for translation into protein.

Another embodiment is a method of preparing a protein compositioncontaining growing recombinant host cell comprising a vector thatencodes a protein which includes an amino acid sequence in accordancewith SEQ ID NO:2, under conditions permitting nucleic acid expressionand protein production followed by recovering the protein so produced.The host cell, conditions permitting nucleic acid expression, proteinproduction and recovery, will be known to those of skill in the art, inlight of the present disclosure of the vls gene.

Gene Constructs and DNA Segments

As used herein, the terms “gene” and “DNA segment” are both used torefer to a DNA molecule that has been isolated free of total genomic DNAof a particular species. Therefore, a gene or DNA segment encoding anVMP-like polypeptide refers to a DNA segment that contains sequencesencoding an VMP-like protein, but is isolated away from, or purifiedfree from, total genomic DNA of the species from which the DNA isobtained. Included within the term “DNA segment”, are DNA segments andsmaller fragments of such segments, and also recombinant vectors,including, for example, plasmids, cosmids, phage, retroviruses,adenoviruses, and the like.

The term “gene” is used for simplicity to refer to a functional proteinor peptide encoding unit. As will be understood by those in the art,this functional term includes both genomic sequences and cDNA sequences.“Isolated substantially away from other coding sequences” means that thegene of interest, in this case, a VMP-like protein encoding gene, formsthe significant part of the coding region of the DNA segment, and thatthe DNA segment does not contain large portions of naturally-occurringcoding DNA, such as large chromosomal fragments or other functionalgenes or cDNA coding regions. Of course, this refers to the DNA segmentas originally isolated, and does not exclude genes or coding regions,such as sequences encoding leader peptides or targeting sequences, lateradded to the segment by the hand of man.

Recombinant Vectors Expressing VMP-like Proteins

A particular aspect of this invention provides novel ways in which toutilize VMP-encoding DNA segments and recombinant vectors comprising vlsDNA segments. As is well known to those of skill in the art, many suchvectors are readily available, one particular detailed example of asuitable vector for expression in mammalian cells is that described inU.S. Pat. No. 5,168,050, incorporated herein by reference. However,there is no requirement that a highly purified vector be used, so longas the coding segment employed encodes a VMP-like protein and does notinclude any coding or regulatory sequences that would have an adverseeffect on cells. Therefore, it will also be understood that usefulnucleic acid sequences may include additional residues, such asadditional non-coding sequences flanking either of the 5′ or 3′ portionsof the coding including, for example, promoter regions, or may includevarious internal sequences, i.e., introns, which are known to occurwithin genes.

After identifying an appropriate VMP-encoding gene or DNA molecule, itmay be inserted into any one of the many vectors currently known in theart, so that it will direct the expression and production of theVMP-like protein when incorporated into a host cell. In a recombinantexpression vector, the coding portion of the DNA segment is positionedunder the control of a promoter. The promoter may be in the form of thepromoter which is naturally associated with a VMP-encoding gene, as maybe obtained by isolating the 5′ non-coding sequences located upstream ofthe coding segment or exon, for example, using recombinant cloningand/or PCR™ technology, in connection with the compositions disclosedherein.

In certain embodiments, it is contemplated that particular advantageswill be gained by positioning the VMP-encoding DNA segment under thecontrol of a recombinant, or heterologous, promoter. As used herein, arecombinant or heterologous promoter is intended to refer to a promoterthat is not normally associated with a vls gene in its naturalenvironment. Such promoters may include those normally associated withother borrelia-inhibitory polypeptide genes, and/or promoters isolatedfrom any other bacterial, viral, eukaryotic, or mammalian cell.Naturally, it will be important to employ a promoter that effectivelydirects the expression of the DNA segment in the particular cellcontaining the vector comprising a vls gene or gene segment.

The use of recombinant promoters to achieve protein expression isgenerally known to those of skill in the art of molecular biology, forexample, see Sambrook et al., (1989). The promoters employed may beconstitutive, or inducible, and can be used under the appropriateconditions to direct high level or regulated expression of theintroduced DNA segment. The currently preferred promoters are those suchas CMV, RSV LTR, the SV40 promoter alone, and the SV40 promoter incombination with the SV40 enhancer.

Methods of DNA Transfection

Technology for introduction of DNA into cells is well-known to those ofskill in the art. Five general methods for delivering a gene into cellshave been described: (1) chemical methods (Graham and VanDerEb, 1973);(2) physical methods such as microinjection (Capecchi, 1980),electroporation (Wong and Neumann, 1982; Fromm et al., 1985) and thegene gun (Yang et al., 1990); (3) viral vectors (Clapp, 1993; Danos andHeard, 1992; Eglitis and Anderson, 1988), (4) receptor-mediatedmechanisms (Wu et al., 1991; Curiel et al., 1991; Wagner et al., 1992);and (5) direct injection of purified DNA into human or animals.

Liposomes and Nanocapsules

The formation and use of liposomes is generally known to those of skillin the art (see for example, Couvreur et al., 1991 which describes theuse of liposomes and nanocapsules in the targeted antibiotic therapy ofintracellular bacterial infections and diseases). Recently, liposomeswere developed with improved serum stability and circulation half-times(Gabizon and Papahadjopoulos, 1988; Allen and Choun, 1987). Thefollowing is a brief description of these DNA delivery modes.

Nanocapsules can generally entrap compounds in a stable and reproducibleway (Henry-Michelland et al., 1987). To avoid side effects due tointracellular polymeric overloading, such ultrafine particles (sizedaround 0.1 mm) should be designed using polymers able to be degraded invivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meetthese requirements are contemplated for use in the present invention,and such particles may be are easily made, as described (Couvreur etal., 1984; 1988).

Liposomes are formed from phospholipids that are dispersed in an aqueousmedium and spontaneously form multilamellar concentric bilayer vesicles(also termed multilamellar vesicles (MLVs). MLVs generally havediameters ranging from 25 mm to 4 mm. Sonication of MLVs results in theformation of small unilamellar vesicles (SUVs) with diameters in therange of 200 to 500 Å, containing an aqueous solution in the core.

In addition to the teachings of Couvreur et al. (1991), the followinginformation may be utilized in generating liposomal formulations.Phospholipids can form a variety of structures other than liposomes whendispersed in water, depending on the molar ratio of lipid to water. Atlow ratios the liposome is the preferred structure. The physicalcharacteristics of liposomes depend on pH, ionic strength and thepresence of divalent cations. Liposomes can show low permeability toionic and polar substances, but at elevated temperatures undergo a phasetransition which markedly alters their permeability. The phasetransition involves a change from a closely packed, ordered structure,known as the gel state, to a loosely packed, less-ordered structure,known as the fluid state. This occurs at a characteristicphase-transition temperature and results in an increase in permeabilityto ions, sugars and drugs.

Liposomes interact with cells via four different mechanisms: Endocytosisby phagocytic cells of the reticuloendothelial system such asmacrophages and neutrophils; adsorption to the cell surface, either bynonspecific weak hydrophobic or electrostatic forces, or by specificinteractions with cell-surface components; fusion with the plasma cellmembrane by insertion of the lipid bilayer of the liposome into theplasma membrane, with simultaneous release of liposomal contents intothe cytoplasm; and by transfer of liposomal lipids to cellular orsubcellular membranes, or vice versa, without any association of theliposome contents. It often is difficult to determine which mechanism isoperative and more than one may operate at the same time.

Expression of VMP-like Proteins

For the expression of VMP-like proteins, once a suitable (full-length ifdesired) clone or clones have been obtained, whether they be cDNA basedor genomic, one may proceed to prepare an expression system for therecombinant preparation of VMP-like proteins. The engineering of DNAsegment(s) for expression in a prokaryotic or eukaryotic system may beperformed by techniques generally known to those of skill in recombinantexpression. It is believed that virtually any expression system may beemployed in the expression of VMP-like proteins.

VMP-like proteins may be successfully expressed in eukaryotic expressionsystems, however, it is also envisioned that bacterial expressionsystems may be preferred for the preparation of VMP-like proteins forall purposes. The cDNA for VMP-like proteins may be separately expressedin bacterial systems with the encoded proteins being expressed asfusions with b-galactosidase, ubiquitin, Schistosoma japonicumglutathione S-transferase, green fluorescent protein and the like. It isbelieved that bacterial expression will ultimately have advantages overeukaryotic expression in terms of ease of use and quantity of materialsobtained thereby.

It is proposed that transformation of host cells with DNA segmentsencoding VMP-like proteins will provide a convenient means for obtainingVMP-like peptides. Both cDNA and genomic sequences are suitable foreukaryotic expression, as the host cell will, of course, process thegenomic transcripts to yield functional mRNA for translation intoprotein.

It is similarly believed that almost any eukaryotic expression systemmay be utilized for the expression of VMP-like proteins, e.g.,baculovirus-based, glutamine synthase-based or dihydrofolatereductase-based systems could be employed. However, in preferredembodiments, it is contemplated that plasmid vectors incorporating anorigin of replication and an efficient eukaryotic promoter, asexemplified by the eukaryotic vectors of the pCMV series, such as pCMV5,will be of most use.

For expression in this manner, one would position the coding sequencesadjacent to and under the control of the promoter. It is understood inthe art that to bring a coding sequence under the control of such apromoter, one positions the 5′ end of the transcription initiation siteof the transcriptional reading frame of the protein between about 1 andabout 50 nucleotides “downstream” of (i.e., 3′ of) the chosen promoter.

Where eukaryotic expression is contemplated , one will also typicallydesire to incorporate into the transcriptional unit which includesVMP-like protein, an appropriate polyadenylation site (e.g.,5′-AATAAA-3′) if one was not contained within the original clonedsegment. Typically, the poly A addition site is placed about 30 to 2000nucleotides “downstream” of the termination site of the protein at aposition prior to transcription termination.

Translational enhancers may also be incorporated as par of the vectorDNA. Thus the DNA constructs of the present invention should alsopreferable contain one or more 5′ non-translated leader sequences whichmay serve to enhance expression of the gene products from the resultingmRNA transcripts. Such sequences may be derived from the promoterselected to express the gene or can be specifically modified to increasetranslation of the RNA. Such regions may also be obtained from viralRNAs, from suitable eukaryotic genes, or from a synthetic gene sequence(Griffiths, et al., 1993).

Such “enhancer” sequences may be desirable to increase or alter thetranslational efficiency of the resultant mRNA. The present invention isnot limited to constructs where the enhancer is derived from the native5′-nontranslated promoter sequence, but may also include non-translatedleader sequences derived from other non-related promoters such as otherenhancer transcriptional activators or genes.

It is contemplated that virtually any of the commonly employed hostcells can be used in connection with the expression of VMPs inaccordance herewith. Examples include cell lines typically employed foreukaryotic expression such as 239, AtT-20, HepG2, VERO, HeLa, CHO, WI38, BHK, COS-7, RIN and MDCK cell lines.

It is contemplated that VMP-like protein may be “overexpressed” , i.e.,expressed in increased levels relative to its natural expression inborrelia cells, or even relative to the expression of other proteins ina recombinant host cell containing VMP-encoding DNA segments. Suchoverexpression may be assessed by a variety of methods, includingradio-labeling and/or protein purification. However, simple and directmethods are preferred, for example, those involving SDS/PAGE and proteinstaining or Western blotting, followed by quantitative analyses, such asdensitometric scanning of the resultant gel or blot. A specific increasein the level of the recombinant protein or peptide in comparison to thelevel in natural VMP-producing animal cells is indicative ofoverexpression, as is a relative abundance of the specific protein inrelation to the other proteins produced by the host cell and, e.g.,visible on a gel.

As used herein, the term “engineered” or “recombinant” cell is intendedto refer to a cell into which a recombinant gene, such as a geneencoding a VMP peptide has been introduced. Therefore, engineered cellsare distinguishable from naturally occurring cells which do not containa recombinantly introduced gene. Engineered cells are thus cells havinga gene or genes introduced through the hand of man. Recombinantlyintroduced genes will either be in the form of a cDNA gene (i.e., theywill not contain introns), a copy of a genomic gene, or will includegenes positioned adjacent to a promoter not naturally associated withthe particular introduced gene.

It will be understood that recombinant VMPs may differ from naturallyproduced VMP in certain ways. In particular, the degree ofpost-translational modifications, such as, for example, lipidation,glycosylation and phosphorylation may be different between therecombinant VMP and the VMP polypeptide purified from a natural source,such as Borrelia.

After identifying an appropriate DNA molecule by any or a combination ofmeans as described above, the DNA may then be inserted into any one ofthe many vectors currently known in the art and transferred to aprokaryotic or eukaryotic host cell where it will direct the expressionand production of the so-called “recombinant” version of the protein.The recombinant host cell may be selected from a group consisting of S.mutans, E. coli, S. cerevisae. Bacillus sp., Lactococci sp., Enterococcisp., or Salmonella sp. In certain preferred embodiments, the recombinanthost cell will have a recA phenotype.

Where the introduction of a recombinant version of one or more of theforegoing genes is required, it will be important to introduce the genesuch that it is under the control of a promoter that effectively directsthe expression of the gene in the cell type chosen for engineering. Ingeneral, one will desire to employ a promoter that allows constitutive(constant) expression of the gene of interest. The use of theseconstitutive promoters will ensure a high constant level of expressionof the introduced genes. The level of expression from the introducedgenes of interest can vary in different clones, probably as a functionof the site of insertion of the recombinant gene in the chromosomal DNA.Thus, the level of expression of a particular recombinant gene can bechosen by evaluating different clones derived from each transfectionstudy; once that line is chosen, the constitutive promoter ensures thatthe desired level of expression is permanently maintained. It may alsobe possible to use promoters that are subject to regulation, such asthose regulated by the presence of lactose analog or by the expressionof bacteriophage T7 DNA polymerase.

Recombinant VMP-like Polypeptides

Recombinant versions of a protein or polypeptide are deemed as part ofthe present invention. Thus one may, using techniques familiar to thoseskilled in the art, express a recombinant version of the polypeptide ina recombinant cell to obtain the polypeptide from such cells. Thetechniques are based on cloning of a DNA molecule encoding thepolypeptide from a DNA library, that is, on obtaining a specific DNAmolecule distinct from other DNAs. One may, for example, clone a cDNAmolecule, or clone genomic DNA. Techniques such as these would also beappropriate for the production of the VMP-like polypeptides inaccordance with the present invention.

Enhanced Production of VMP-like Proteins

Potential problems with VMP-like proteins isolated from natural sourcesare low yields and extensive purification processes. An aspect of thepresent invention is the enhanced production of VMP-like proteins byrecombinant methodologies in a bacterial host, employing DNA constructsto transform Gram-positive or Gram-negative bacterial cells. Forexample, the use of Escherichia coli expression systems is well known tothose of skill in the art, as is the use of other bacterial species suchas Bacillus subtilis or Streptococcus sanguis.

Further aspects of the invention include high expression vectorsincorporating DNA encoding novel vls, combinatorial segments and itsvariants. It is contemplated that vectors providing enhanced expressionof VMP in other systems such as S. mutans will also be obtainable. Whereit is desirable, modifications of the physical properties of VMP may besought to increase its solubility or expression in liquid culture. Thevls locus may be placed under control of a high expression promoter orthe components of the expression system altered to enhance expression.

In further embodiments, the DNA encoding the VMP-like proteins of thepresent invention allows for the large scale production and isolation ofVMP-like polypeptides. This can be accomplished by directing theexpression of the VMP-like polypeptide by cloning the DNA encoding theVMP-like polypeptide into a suitable expression vector. Such anexpression vector may then be transformed into a host cell that is ableto produce the VMP-like proteins. The VMP-like protein may then bepurified, e.g., by means provided for in this disclosure and utilized ina biologically active form. Non-biologically active recombinant VMP-likeproteins may also have utility, e.g., as an immunogen to prepare anti-VMantibodies.

Gene Immunization

Adenovirus is particularly suitable for use as a gene transfer vectorbecause of its mid-sized DNA genome, ease of manipulation, high titer,wide target-cell range, and high infectivity. The roughly 36 kB viralgenome is bounded by 100-200 base pair (bp) inverted terminal repeats(ITR), in which are contained cis-acting elements necessary for viralDNA replication and packaging. The early (E) and late (L) regions of thegenome that contain different transcription units are divided by theonset of viral DNA replication.

The E1 region (E1A and E1B) encodes proteins responsible for theregulation of transcription of the viral genome and a few cellulargenes. The expression of the E2 region (E2A and E2B) results in thesynthesis of the proteins for viral DNA replication. These proteins areinvolved in DNA replication, late gene expression, and host cell shutoff (Renan, 1990). The products of the late genes (L1, L2, L3, L4 andL5), including the majority of the viral capsid proteins, are expressedonly after significant processing of a single primary transcript issuedby the major late promoter (MLP). The MLP (located at 16.8 map units) isparticularly efficient during the late phase of infection, and all themRNAs issued from this promoter possess a 5′ tripartite leader (TL)sequence which makes them preferred mRNAs for translation.

In order for adenovirus to be optimized for gene therapy, it isnecessary to maximize the carrying capacity so that large segments ofDNA can be included. It also is very desirable to reduce the toxicityand immunologic reaction associated with certain adenoviral products.

The large displacement of DNA is possible because the cis elementsrequired for viral DNA replication all are localized in the invertedterminal repeats (ITR) (100-200 bp) at either end of the linear viralgenome. Plasmids containing ITR's can replicate in the presence of anon-defective adenovirus (Hay et al., 1984). Therefore, inclusion ofthese elements in an adenoviral vector should permit replication.

In addition, the packaging signal for viral encapsidation is localizedbetween 194-385 bp (0.5-1.1 map units) at the left end of the viralgenome (Hearing et al., 1987). This signal mimics the proteinrecognition site in bacteriophage λ DNA where a specific sequence closeto the left end, but outside the cohesive end sequence, mediates thebinding to proteins that are required for insertion of the DNA into thehead structure. E1 substitution vectors of Ad have demonstrated that a450 bp (0-1.25 map units) fragment at the left end of the viral genomecould direct packaging in 293 cells (Levrero et al., 1991).

It has been shown that certain regions of the adenoviral genome can beincorporated into the genome of mammalian cells and the genes encodedthereby expressed. These cell lines are capable of supporting thereplication of an adenoviral vector that is deficient in the adenoviralfunction encoded by the cell line. There also have been reports ofcomplementation of replication deficient adenoviral vectors by “helping”vectors, e.g., wild-type virus or conditionally defective mutants.

VMP-like Variants

VMP-like related proteins and functional variants are also consideredpart of the invention. Thus it is expected that truncated and mutatedversions of VMP-like protein (SEQ ID NO:2) will afford more convenientand effective forms of VMP for treatment regimens. Thus, any functionalversion of SEQ ID NO:2, such as truncated species or homologs, andmutated versions of VMP-like protein are considered as part of theinvention.

Mutagenized recombinant VMPs may have increased potency and prolonged invivo half-life, thereby offering more effective long-term treatments.Novel VMPs thus may be obtained by modifications to the vls gene, (suchas by site-specific mutagenesis).

Additionally, the 15 silent vls cassettes of B. burgdorferi may berecombined in numerous combinations, providing for example a cocktail ofpeptide compositions for use as immunogens and to develop vaccines foruse in Lyme disease and related conditions.

Pharmaceutical Compositions

Pharmaceutical compositions prepared in accordance with the presentinvention find use in preventing or ameliorating conditions associatedwith Borrelia infections, particularly Lyme disease. Such methodsgenerally involve administering a pharmaceutical composition comprisingan effective amount of a VMP-like antigen, such as SEQ ID NO:2 orvarious epitopes thereof. Other exemplary compositions may include aneffective amount of either a VMP-like variant or a VMP-like encodingnucleic acid composition. Such compositions may also be used to generatean immune response in an animal in such cases where it may be desirableto block the effect of a naturally produced VMP-like protein.

Also included as part of the present invention therefore are novelcompositions comprising nucleic acids which encode a VMP-like protein.It will, of course, be understood that one or more than one gene may beused in the methods and compositions of the invention. The nucleic aciddelivery methods may thus entail the administration of one, two, three,or more, homologous VMP-encoding genes. The maximum number of genes thatmay be applied is limited only by practical considerations, such as theeffort involved in simultaneously preparing a large number of geneconstructs or even the possibility of eliciting an adverse cytotoxiceffect.

The particular combination of genes may be two or more distinct genes;or it may be such that a vls gene is combined with another gene and/oranother protein, cofactor or other biomolecule; a cytokine gene may evenbe combined with a gene encoding a cell surface receptor capable ofinteracting with the polypeptide product of the first gene.

In using multiple genes, they may be combined on a single geneticconstruct under control of one or more promoters, or they may beprepared as separate constructs of the same or different types. Thus, analmost endless combination of different genes and genetic constructs maybe employed. Certain gene combinations may be designed to, or their usemay otherwise result in, achieving synergistic effects in affordingprotection against Borrelia and or stimulation of an immune response.Any and all such combinations are intended to fall within the scope ofthe present invention. Indeed, many synergistic effects have beendescribed in the scientific literature, so that one of ordinary skill inthe art would readily be able to identify likely synergistic genecombinations, or even gene-protein combinations.

It will also be understood that, if desired, the nucleic acid segment orgene encoding a VMP-like protein could be administered in combinationwith further agents, such as, e.g., proteins or polypeptides or variouspharmaceutically active agents. So long as the composition comprises avls gene, there is virtually no limit to other components which may alsobe included, given that the additional agents do not cause a significantadverse effect upon contact with the target cells or host tissues. Thenucleic acids may thus be delivered along with various other agents asrequired in the particular instance.

Kits

Therapeutic kits comprising VMP-like peptides or VMP-encoding nucleicacid segments comprise another aspect of the present invention. Suchkits will generally contain, in suitable container means, apharmaceutically acceptable formulation of a VMP-like peptide or aVMP-encoding nucleic acid composition. The kit may have a singlecontainer means that contains the VMP composition or it may havedistinct container means for the VMP composition and other reagentswhich may be included within such kits.

The components of the kit may be provided as liquid solution(s), or asdried powder(s). When the components are provided in a liquid solution,the liquid solution is an aqueous solution, with a sterile aqueoussolution being particularly preferred. When reagents or components areprovided as a dry powder, the powder can be reconstituted by theaddition of a suitable solvent. It is envisioned that the solvent mayalso be provided in another container means.

Kits may also comprise reagents for detecting VMP-like polypeptides,such as required for immunoassay. The immunodetection reagent willtypically comprise a label associated with the antibody or antigen, orassociated with a secondary binding ligand. Exemplary ligands mightinclude a secondary antibody directed against the first antibody orantigen or a biotin or avidin (or streptavidin) ligand having anassociated label. Of course, a number of exemplary labels are known inthe art and all such labels may be employed in connection with thepresent invention. The kits may contain antibody-label conjugates eitherin fully conjugated form, in the form of intermediates, or as separatemoieties to be conjugated by the user of the kit.

The container means will generally include at least one vial, test tube,flask, bottle, syringe or other container means, into which the antigenor antibody may be placed, and preferably suitably aliquoted. Where asecond binding ligand is provided, the kit will also generally contain asecond vial or other container into which this ligand or antibody may beplaced. The kits of the present invention will also typically include ameans for containing the antibody, antigen, and reagent containers inclose confinement for commercial sale. Such containers may includeinjection or blow-molded plastic containers into which the desired vialsare retained.

VMP Antibodies

In another aspect, the present invention contemplates an antibody thatis immunoreactive with a polypeptide of the invention. An antibody canbe a polyclonal or a monoclonal antibody. In a preferred embodiment, anantibody is a monoclonal antibody. Means for preparing andcharacterizing antibodies are well known in the art (See, e.g., Howelland Lane, 1988).

Briefly, a polyclonal antibody is prepared by immunizing an animal withan immunogen comprising a polypeptide of the present invention andcollecting antisera from that immunized animal. A wide range of animalspecies can be used for the production of antisera. Typically an animalused for production of anti-antisera is a rabbit, a mouse, a rat, ahamster or a guinea pig. Because of the relatively large blood volume ofrabbits, a rabbit is a preferred choice for production of polyclonalantibodies.

Antibodies, both polyclonal and monoclonal, specific for VMP-likepolypeptides and particularly those represented by SEQ ID NO:2, variantsand epitopes thereof, may be prepared using conventional immunizationtechniques, as will be generally known to those of skill in the art. Acomposition containing antigenic epitopes of VMP can be used to immunizeone or more experimental animals, such as a rabbit or mouse, which willthen proceed to produce specific antibodies against vls expression andsilent regions. Polyclonal antisera may be obtained, after allowing timefor antibody generation, simply by bleeding the animal and preparingserum samples from the whole blood.

To obtain monoclonal antibodies, one would also initially immunize anexperimental animal, often preferably a mouse, with a VMP composition.One would then, after a period of time sufficient to allow antibodygeneration, obtain a population of spleen or lymph cells from theanimal. The spleen or lymph cells can then be fused with cell lines,such as human or mouse myeloma strains, to produce antibody-secretinghybridomas. These hybridomas may be isolated to obtain individual cloneswhich can then be screened for production of antibody to the desired VMPpeptide.

Following immunization, spleen cells are removed and fused, using astandard fusion protocol with plasmacytoma cells to produce hybridomassecreting monoclonal antibodies against VMP. Hybridomas which producemonoclonal antibodies to the selected antigens are identified usingstandard techniques, such as ELISA and Western blot methods. Hybridomaclones can then be cultured in liquid media and the culture supernatantspurified to provide the VMP-specific monoclonal antibodies.

It is proposed that the monoclonal antibodies of the present inventionwill find useful application in standard immunochemical procedures, suchas ELISA and Western blot methods, as well as other procedures which mayutilize antibody specific to VMP epitopes.

Additionally, it is proposed that monoclonal antibodies specific to theparticular polypeptide may be utilized in other useful applications. Forexample, their use in immunoabsorbent protocols may be useful inpurifying native or recombinant VMP species or variants thereof.

In general, both poly- and monoclonal antibodies against VMP may be usedin a variety of embodiments. For example, they may be employed inantibody cloning protocols to obtain cDNAs or genes encoding VMP orrelated proteins. They may also be used in inhibition studies to analyzethe effects of VP in cells or animals. Anti-VMP antibodies will also beuseful in immunolocalization studies to analyze the distribution of VMPpeptides during various cellular events, for example, to determine thecellular or tissue-specific distribution of the VP peptide underdifferent physiological conditions. A particularly useful application ofsuch antibodies is in purifying native or recombinant VMP, for example,using an antibody affinity column. The operation of all suchimmunological techniques will be known to those of skill in the art inlight of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A. Correlation of infectivity of B. burgdorferi B31 clones 5A1through 5A10 with presence of a 28-kb linear plasmid (pBB2La). Plasmidprofiles of B31 clones as determined by pulse-field gel electrophoresisand ethidium bromide staining. Low-(−) and high-(+) infectivity B31clones have a virtually identical plasmid banding pattern by thismethod.

FIG. 1B. Correlation of infectivity of B. burgdorferi B31 clones 5A1through 5A10 with presence of a 28-kb linear plasmid (pBB28La).Hybridization of a DNA blot of the gel shown in FIG. 1A with the pJRZ53probe. The probe hybridized specifically with a 28-kb plasmid present inall 5 high-infectivity clones but in only 1 of 4 low-infectivity clones.Molecular sizes of the standards are indicated in kilobases, and anasterisk marks the location of pBB28La in the ethidium bromide-stainedplasmid profile.

FIG. 2A. Structure of the vls locus of B. burgdorferi clone B31-5A3.Diagrammatic illustration of the overall arrangement of the vls locus inB. burgdorferi plasmid pBB28La. Distances from the left telomere areindicated in kb, and the locations of the subtractive hybridizationclone pJRZ53 and the λDASH-Bb12 inserts are shown.

FIG. 2B. Structure of the vls locus of B. burgdorferi clone B31-5A3.Structure of vlsE.

FIG. 2C. Structure of the vls locus of B. burgdorferi clone B31-5A3.Structure of vlsE. Nucleotide and predicted amino acid sequences of theallele vlsE1 of the B. burgdorferi B31-5A3 vlsE gene. The predicted −10and −35 promoter sequences, the putative ribosome binding site (RBS),and primers used for PCR™ and RT-PCR™ are marked. FIG. 2C shows thenucleotide and amino acid sequences of SEQ ID NO:1 and SEQ ID NO:2,respectively.

FIG. 3A. Sequence similarity of the predicted VlsE sequence (alleleVlsE1) with the variable major proteins (Vmps) of B. hermsii and thepredicted amino acid sequences of the silent vls cassettes. Alignment ofthe predicted amino acid sequence of VlsE (allele VlsE1) with that ofVmp17 (GenBank entry L04788). Identical amino acid residues areindicated by vertical lines (|) and similar residues are marked withcolons (:) and periods (.). FIG. 3A upper sequence shows the amino acidsequences of SEQ ID NO:13, while the bottom sequence corresponds to SEQID NO:14.

FIG. 3B. Sequence similarity of the predicted VlsE sequence (alleleVlsE1) with the variable major proteins (Vmps) of B. hermsii and thepredicted amino acid sequences of the silent vls cassettes. Alignment ofthe deduced peptide sequences of 16 vls cassettes. Residues identical tothe VlsE cassette region (vls1) of B. burgdorferi are marked as dashes(−); similar amino acids are shown in lower case. Gaps and the predictedstop codons are indicated by dots (.) and asterisk (*), respectively.Variable regions VR-I through VR-VI are shaded. Vls1 corresponds to SEQID NO:15, Vls2 corresponds to SEQ ID NO:16, Vls3 corresponds to SEQ IDNO:17, Vls4 corresponds to SEQ ID NO:18, Vls5 corresponds to SEQ IDNO:19, Vls6 corresponds to SEQ ID NO:20, Vls7 corresponds to SEQ IDNO:21, Vls8 corresponds to SEQ ID NO:22, Vls9 corresponds to SEQ IDNO:23, Vls10 corresponds to SEQ ID NO:24, Vls11 corresponds to SEQ IDNO:25, Vls12 corresponds to SEQ ID NO:26 and Vls13 corresponds to SEQ IDNO:27 Vls14 corresponds to SEQ ID NO:28, Vls15 corresponds to SEQ IDNO:29 and Vls16 corresponds to SEQ ID NO:30.

FIG. 4A. Surface localization of vlsE, as indicated by treatment ofintact B. burgdorferi with proteinase K. Freshly cultured B. burgdorferiB31 clone 5A3 cells were incubated with (+) or without (−) proteinase Kat room temperature for 10 min. The proteins of the washed organismswere then separated by SDS-PAGE. The protein blots were reacted withantiserum against the GST-vls1 fusion protein;

FIG. 4B. Surface localization of VlsE, as indicated by treatment ofintact B. burgdorferi with proteinase K. Freshly cultured B. burgdorferiB31 clone 5A3 cells were incubated with (+) or without (−) proteinase Kat room temperature for 10 min. The proteins of the washed organismswere then separated by SDS-PAGE The protein blots were reacted withantiserum against B. burgdorferi B31 OspD.

FIG. 4C. Surface localization of VlsE, as indicated by treatment ofintact B. burgdorferi with proteinase K. Freshly cultured B. burgdorferiB31 clone 5A3 cells were incubated with (+) or without (−) proteinase Kat room temperature for 10 min. The proteins of the washed organismswere then separated by SDS-PAGE. The protein blots were reacted withmonoclonal antibody H9724 against the B. burgdorferi flagellin (Fla).

FIG. 5A. Changes in deduced amino acid sequences of VlsE occurringduring infection of C3H/HeN mice with B. burgdorferi B31-5A3. Flow chartof the overall experimental design.

FIG. 5B. Changes in deduced amino acid sequences of VlsE occurringduring infection of C3H/HeN mice with B. burgdorferi B31-5A3. Amino acidsequence alignment of the vlsE alleles in one clonal population fromeach of 11 different isolates. VlsE1corresponds to SEQ ID NO:31, M1e4Acorresponds to SEQ ID NO:32, M1b4A corresponds to SEQ ID NO:33, M2b4Acorresponds to SEQ ID NO:34, M3e4A corresponds to SEQ ID NO:35, M3b4Acorresponds to SEQ ID NO:36, M4e4A corresponds to SEQ ID NO:37, M4b4Acorresponds to SEQ ID NO:38, M5e4A corresponds to SEQ ID NO:39, M6b4Acorresponds to SEQ ID NO:40, M7b4A corresponds to SEQ ID NO:41 and M8e4Acorresponds to SEQ ID NO:42.

FIG. 5C. Changes in deduced amino acid sequences of vlsE occurringduring infection of C3H/HeN mice with B. burgdorferi B31-5A3 Amino acidsequence alignment of the vlsE alleles in 5 clonal populations from asingle ear isolate. In FIG. 5B and FIG. 5C, the deduced amino acidsequences of the mouse isolates were compared with those of theinoculating clone (VlsE1); similarity to this sequence is depicted asdescribed in FIG. 3B. Amino acid residues (EGAIK) encoded by the 17-bpdirect repeat are highlighted to indicate the boundaries of the vlscassette. VlsE1 corresponds to SEQ ID NO:43, M1e4A corresponds to SEQ IDNO:44, M1e4B corresponds to SEQ ID NO:45, M1e4C corresponds to SEQ IDNO:46, M1e4D corresponds to SEQ ID NO:47 and M1e4E corresponds to SEQ IDNO:48.

FIG. 6A. Altered VlsE antigenicity of B. burgdorferi clones (m1e4Athrough m8e4A) isolated from C3H/HeN mice 4 weeks post infection. Theantigenic reactivties of 9 clones isolated from mice (lanes 109) werecompared with those of the parental clone B31-5A3 used for mouseinoculation (lane 11) and the low-infectivity clone B31-5A2 (lane 10),which lacks the plasmid encoding VlsE. Two identical SDS-PAGE westernblots were reacted with monoclonal antibody H9724 directed against theB. burgdorferi flagellin protein (Fla) as a positive control.

FIG. 6B. Altered VlsE antigenicity of B. burgdorferi clones (m1e4Athrough m8e4A) isolated from C3H/HeN mice 4 weeks post infection andantiserum against the GST-vls1 fusion protein. Antiserum against theGST-vls1 fusion protein. Prolonged exposures of the immunoblot againstthe GST-vls1 futions protein indicated the presence of weakly reactivebands in all 9 mouse isolates. The relative locations of proteinstandards are indicated.

FIG. 6C. Reactivity of serum antibodies from a representative Musmusculus C3H/HeN mouse with vlsE. An immunoblot of B. burgdorferiproteins from the strains indicated and the GST-vls1 fusion protein werereacted with serum from mouse 1 obtained 28 days after needleinoculation with 10⁵ B. burgdorferi B31, clone 5A3.

FIG. 6D. Reactivity of serum antibodies from a representative Musmusculus C3H/HeN mouse with VlsE. An immunoblot of B. burgdorferiproteins from the strains indicated and the GST-vls1 fusion protein werereacted with serum from a Peromyscus leukopcus mouse infected with B.burgdorferi B31 via tick-bite. The protein bands corresponding to VlsEand the SGT-Vls1 fusion protein (as determined by reactivity withanti-GST-Vls1 antiserum; data not shown) are indicated by arrows. Therelative locations of protein standards are shown in kilodaltons.

FIG. 6E. Reactivity of serum antibodies from a representative Musmusculus C3H/HeN mouse with VlsE. An immunoblot of B burgdorferi[proteins from the strains indicated and the GST-vls1 fusion proteinwere reacted with serum from an early stage Lyme disease patient. Theprotein bands corresponding to VlsE and the GST-vls1 fusion protein (asdetermined by reactivity with anti-GST-vls1 antiserum; data not shown)are indicated by arrows. The relative locations of protein standards areshown in kilodaltons.

FIG. 7. Proposed model for genetic and antigenic variation at the vlslocus. Recombination of segments of the silent vls cassettes vls7 andvls4 into the vls1 cassette of B. burgdorferi B31-5A3 vlsE gene isshown. A series of similar recombination events would generate uniquevlsE alleles consisting of a mosaic of segments from several differentsilent vls cassettes.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present work discloses the identification and characterization of anelaborate genetic system in the Lyme disease spirochete Borreliaburgdorferi that promotes extensive antigenic variation of asurface-exposed lipoprotein, VlsE. A 28-kilobase linear plasmid of B.burgdorferi B31 (pBB28La) was found to contain a vmp-like sequence (vls)locus that closely resembles the variable major protein (vmp) system forantigenic variation of relapsing fever organisms. Portions of several ofthe 15 non-expressed (silent) vls cassette sequences located upstream ofvlsE recombined into the central vlsE cassette region during infectionof C3H/HeN mice, resulting in antigenic variation of the expressedlipoprotein. The resulting combinatorial variation will potentiallyproduce millions of unique antigenic variants and thereby contribute toimmune evasion, long-term survival, and pathogenesis in the mammalianhost.

An infectivity-associated 28-kb linear plasmid, pBB28La. in B.burgdorferi B31 by subtractive hybridization has been identified. DNAsequence analysis of cloned fragments from this plasmid revealed the vlslocus consisting of 15 silent vls cassettes and an expressed VlsE gene.Subsequent infection studies demonstrated that promiscuous recombinationoccurs at the vlsE site in C3H/HeN mice. Although the vls locus has beencharacterized thoroughly only in one clonal population of B. burgdorferiB31, Southern hybridization results indicate that this locus is presentin infectious strains of three well-defined Lyme disease Borreliaegenospecies (B. burgdorferi , B. afzelii, and B. garinii), despite theoverall genetic heterogeneity among these organisms (Casjens et al.,1995; Xu and Johnson, 1995).

The vls locus resembles the vmp system of B. hermsii in both sequenceand genetic organization. There is some sequence homology between thesetwo systems, particularly between the vlsE and large vmp genes. This isexemplified by direct sequence comparison between vlsE and vmp17, whichhave homology throughout their predicted amino acid sequences (FIG. 3A).The vlsE and silent vls cassettes also have a closer degree of homologyto small vmps and B. burgdorferi ospC genes. Additionally, both the vlsand vmp systems have a single expression site encoding asurface-localized lipoprotein, as well as multiple unexpressed sequences(Plasterk et al., 1985; Barbour et al., 1991a). Finally, the expressionsites for both systems are located near one of the telomeres of theirrespective linear plasmids (Kitten and Barbour, 1990; Barbour et al.,1991b). These observations suggest that the vls locus may provide theLyme disease Borreliae with the capability of antigenic variationanalogous to the vmp system of B. hermsii (Barbour, 1993). The abovesimilarities also indicate that the VlsE gene, silent vls cassettes, andlarge vmp genes of relapsing fever organisms, all evolved from a commonancestral gene. Their relatively high G+C compositions (e.g. 45% forVlsE and 37% for vmp17) when compared with Borrelia G+C content (˜28%)are also consistent with this evolutionary relationship, and furthersuggest the possibility of lateral transfer from other organisms.

There are several differences between the vls and vmp systems. First, B.hermsii possesses at least two vmp-containing linear plasmids (Meier etal., 1985; Plasterk et al., 1985), whereas only one vls-containinglinear plasmid was detected in Lyme disease Borreliae underhybridization conditions (FIGS. 1A and 1B). Second, the silent vmp genesare separated by intergenic noncoding regions and arranged in eitherorientation (Barbour et al., 1991a), but the silent vls cassettes areorganized head-to-tail as a single open reading frame throughout almostthe entire region (FIGS. 2A and 2B). Third, the silent vmp genes lackpromoter sequences, but most encode complete or nearly complete openreading frames with their own ribosome-binding sites (Barbour et al.,1991a). On the other hand, the vls cassettes represent only the centralthird of the expression site. Lastly, each phase of B. hermsii infectionis caused predominantly by organisms expressing a single vmp allele(Meier et al., 1985; Plasterk et al., 1985), whereas a high degree ofvlsE allelic variation occurs among organisms isolated even from a smallear biopsy specimen during B. burgdorferi infection (FIGS. 5A, 5B and5C).

The sequence changes at the vlsE site may result from geneticrecombination with sequences from the silent vls cassettes. Despiteconsiderable sequence variations within the vls region of different vlsEalleles, the sequence examined outside the 17-bp direct repeats remainedunchanged (FIG. 5B and FIG. 5C). Within the vls region, the changes arenot random but are clustered predominantly in six highly variableregions found in 15 silent vls cassettes (FIG. 3B). Nearly all of thesequence variations observed in the mouse isolates are identical toportions of the silent vls cassettes, although the combinations of thesequence variations made each of these alleles unique.

The inventors have shown that B. burgdorferi undergoes an unusual typeof genetic variation (FIG. 7): (i) the vls cassettes contain conservedand variable regions; (ii) the conserved sequences facilitaterecombination between the expressed and silent vls sequences, probablyby a non-reciprocal gene conversion mechanism; (iii) the conserved 17-bpdirect repeat sequences may be involved in alignment of the vlssequences during recombination or in binding of proposed site-specificrecombinase(s); (iv) through multiple recombination events, portions ofthe expression site are replaced by segments from several silent vlscassettes, resulting in a vast array of potential vlsE alleles; and (v)the site-specific mechanism is activated in vivo, resulting in a highrate of recombination. Since both the vlsE and silent vls cassettes arelocated on the same linear plasmid, pBB28La, in B. burgdorferi (FIG.2A), intraplasmic recombination is likely to be involved. However, it isalso possible that interplasmic recombination of multiple copies of thepBB28La plasmid are present in each organism, as shown with thevmp-encoding plasmids of B. hermsii (Kitten and Barbour, 1992).

Genetic variation involved in multi-gene families has been described inseveral other pathogenic microorganisms (Borst and Geaves, 1987; Borstet al., 1995; Donelson, 1995). In the context of combinatorialrecombination, the genetic variation at the vlsE site is similar to thatof the pilin-encoding genes of N. Gonorrhoeae (Seifert and So, 1988).The gonococcal pilus is primarily composed of repeating subunits of an18-kilodalton pilin protein and is required for adherence of thebacterium to a variety of human cells (Swanson and Koomey, 1989). Whilethe complete pilin genes are expressed only at two expression sites(pilE1 and pilE2), multiple silent copies (pilS) containing portions ofthe pilin genes are found over a wide range on the gonococcal chromosome(Haas and Meyer, 1986). Through multiple combinatorial recombinationevents, a single gonococcal clone expressing one pilin stereotype cangive rise to a large number of progeny that express antigenicallydistinctive pilin variants (Meyer et al., 1982; Hagblom et al., 1985;Segal et al., 1986). The recombination between the expression and silentloci occurs predominantly through a non-reciprocal gene conversionmechanism (Haas and Meyer, 1986; Koomey et al., 1987).

The coding sequences of the Neisseria pilin variants are divided intoconstant, semi-variable, and hypervariable regions (Haas and Meyer,1986), which are analogous to the conserved and variable regions of thevls cassettes (FIG. 3B, FIG. 5B and FIG. 5C). The constant regions and aconserved DNA sequence (Sma/Cla repeat) located at the 3′ end of allpilin loci are though to pair the regions involved in recombinationevents (Wainwright et al., 1994). In this context, the 17-bp directrepeats (FIG. 2C) and the conserved regions (FIG. 3B) of the vlscassettes may play a similar role in recombination events. The silentloci of gonococcal pilin genes contain different regions of the completepilin genes (Haas and Meyer, 1986), whereas the silent vls cassettes ofB. burgdorferi represent only the central cassette region of the vlsEgene (FIG. 3B).

Non-reciprocal recombinations also occurs between the expressed and thesilent genes encoding variant surface glycoproteins (Vsgs) in Africantrypanosomes (Donelson, 1995). Based on similarities between the vlslocus and the multi-gene families of the other pathogenicmicroorganisms, it is likely that a unidirectional gene conversionmechanism is also active in the vls locus. However, there is not as yetany data regarding the preservation of the silent vls cassettes, and theexact mechanism of vls recombination remains to be determined.

There is strong evidence that genetic variation at the vls locusgenerates antigenic variation. The prolific recombination at the vlsEsite in C3H/HeN mice supports the possibility of antigenic variation inLyme diseases caused by Borreliae. The decreased reactivity to antibodyagainst the parental vls1 cassette region among the clonal populationsof mouse isolates demonstrates that genetic variation at the vlsE siteresulted in changes in antigenicity of the vlsE variants (FIG. 6B).Finally, C3H/HeN mice infected with B. burgdorferi produced strongantibody responses against the parental VlsE protein, but consistentwith the results obtained with the antibody against the GST-vls1 fusionprotein, the same antisera had decreased reactivities with some of theVlsE variants isolated from mice (FIG. 6C). Since VlsE is asurface-exposed lipoprotein, as indicated by proteinase K digestion(FIGS. 4A and 4B) and [³H]-palmitate radiolabeling studies, thisproposed antigenic variation may allow Lyme disease Borreliae to surviveimmune attack targeted against VlsE.

Variation of B. burgdorferi surface proteins such as VlsE may alsoaffect the organism's virulence and its ability to adapt to differentmicro-environments during infection of the mammalian host. Recentstudies of a Borrelia turicatae mouse infection model that resemblesLyme disease showed that one serotype expressing VmpB exhibited moresevere arthritic manifestations, whereas another expressing VmpA hadmore severe central nervous system involvement (Cadavid et al, 1994).The numbers of Borreliae present in the joints and blood of serotypeB-infected mice were much higher than those of mice infected withserotype A, consistent with a relationship between Vmp serotype anddisease severity (Pennington et al, 1997). Antigenic variation ofNeisseria pilin (Lambden et al., 1980; Rudel et al., 1992; Nassif etal., 1993; Jonsson et al, 1994) and Opa proteins (Kupsch et al, 1993) isknown to affect adherence of the organisms to human leukocytes andepithelial cells.

The importance of the vls-containing plasmid, pBB28La, during infectionis supported by the following evidence: (i) all high-infectivity clonesand strains tested thus far contain the vls-containing plasmid pBB28La,and loss of this plasmid correlates with a decrease in infectivity (FIG.1B); (ii) pBB28La was maintained in all animal isolates tested thus far;and (iii) the vls sequences are preserved among three Lyme diseasegenospecies despite their genetic heterogeneity (Casjens et al., 1995)and diversity in plasmid profiles (Xu and Johnson, 1995). On the otherhand, B. burgdorferi clones with or without plasmid pBB28La showedsimilar growth rates in culture medium. In addition, pBB28La is readilylost during in vitro subcultures as early as passage 5. Therefore,presence of pBB28La appears to have little if any effect on in vitrogrowth, yet has a profound effect on the ability to infect mammalianhost.

VlsE (or, potentially, other genes encoded by pBB28La) appears to haveanother important but undefined function which is unrelated to antigenicvariation. Low-infectivity clones lacking the vls-encoding plasmidpBB28La do not propagate in severe combined immunodeficiency (SCID)mice, indicating that the required factor(s) provides an importantfunction unrelated to evasion of the adaptive immune system. Also, invivo selection against Bb clones lacking pBB28La appears to occur earlyin infection (within the first week), before the adaptive immuneresponse would be expected to exert significant selection pressure.Therefore, it is likely that VlsE plays an important role in some aspectof infection (e.g. colonization, dissemination, adherence,extravasation, evasion of innate immune mechanisms, or nutrientacquisition), and that antigenic variation merely permits surfaceexpression of this protein without leading to elimination of thebacteria by the host's immune response. Retention of this activity wouldrequire that the variation in amino acid sequences would not interferewith the active site(s) of the protein; this requirement may explain theexistence of highly conserved regions at the N- and C-termini and withinthe vls cassette. Sequence variation as a mechanism of maintainingsurface protein function in the face of a hostile immune response may bea strategy common to pathogenic microorganisms.

Antigenic Variation in B. hermsii

A complex antigenic variation mechanism has been characterized inBorrelia hermsii, a relative of B. burgdorferi that causes relapsingfever (Balmelii and Piffatetti, 1996; Barbour, 1993; Donelson, 1995).Surface-exposed lipoproteins called variable major proteins (Vmps) areencoded by homologous genes located in 28- to 32-kb linear plasmids withcovalently closed telomeres (Barbour and Garon, 1987; Kitten andBarbour, 1990). The vmp genes have been subdivided into two groups:small and large (Restrepo et al., 1992). Large vmp genes such as vmp7and vmp17 and small vmp genes such as vmp1 and vmp3 are approximately 1kb and 0.6 kb in size, respectively. Each organism contains both smalland large vmp genes in a unexpressed (silent) form in the so-calledstorage plasmids (Plasterk et al., 1985). Only one vmp gene located nearone of the telomeres of a different plasmid (called the expressionplasmid) is expressed in each organism (Kitten and Barbour, 1990;Barbour et al., 1991a). Antigenic variation occurs when the expressedvmp is replaced completely or partially by one of the silent vmp genesat the telomeric expression site through interplasmic recombination(Meier et al., 1985; Plasterk et al., 1985; Barbour et al., 1991b),intraplasmic recombination (Restrepo et al., 1994), and post-switchrearrangement (Restrepo and Barbour, 1994). The antigenic switch occursspontaneously at a frequency of 10⁻³ to 10⁻⁴ per generation (Stoenner etal., 1982).

Identification of vls

A genetic locus (called vmp-like sequence or vls) has been identifiedand characterized in B. burgdorferi that surprisingly resembles the vmpsystem of B. hermsii. A vls expression site (vlsE) and 15 additionalsilent vls cassettes were identified on a 28-kb linear plasmid(designated pBB28La), The presence of pBB28La correlates with thehigh-infectivity phenotype in B. burgdorferi sensu lato strains tested.vlsE, located near a telomere of pBB28La, encodes a surface-exposedlipoprotein. Examination of ear and blood isolates from C3H/HeN miceinfected 4 weeks previously with B31 clone 5A3 demonstrated theoccurrence of promiscuous recombination at the vlsE site, such that eachof B. burgdorferi clones examined was unique and appeared to haveundergone multiple recombination events with portions of the silent vlscassettes. The resultant VlsE variants exhibited a decreased reactivityto antiserum directed against the parental vls1 cassette region. Thiselaborate genetic system permits combinatorial antigenic variation ofvlsE in the mammalian host, thereby contributing to evasion of theimmune response and long-term survival in the mammalian host.

The present invention illustrates the rapid occurrence of promiscuousrecombination at the vls expression site (vlsE), resulting in acombinatorial form of genetic and antigenic variation at the vlsE site.Antigenic variation at the vls site has been detected using an in vivoselection approach.

The sequence variation appears to lead to significant antigenicvariation. Rabbit antiserum raised against a vls1-GST fusion proteinreacted strongly with the original B. burgdorferi clone (B31 5A3), butdid not react with several of the clones reisolated from mice 4 weekspost infection.

B. burgdorferi induces a site-specific recombination mechanism duringinfection of the mammalian host. The vlsE cassette sequence in each ofthe mouse isolates is unique. At the nucleotide level each vlsE cassetteis comprised of regions identical to several of the silent vlscassettes. This promiscuous recombination of silent vls cassettesegments causes a combinatorial diversity at the VlsE expression site,similar to the diversity possible in the immunoglobulin and Tcell-receptor variable regions. In contrast, antigenic variation inrelapsing fever organisms usually involves replacement of the entiregene at the expression site with one of the ‘silent’ VMP genes.Moreover, a single VMP serotype is predominant during each relapse.

This mechanism of genetic switching appears to be different from anyother antigenic variation mechanism described in bacteria or protozoaand has important implications in Lyme disease. By combining differentregions of the silent vls cassettes, it is possible for many differentVlsE ‘serotypes’ to coexist in the same patient. It may be impossiblefor the host to mount a protective response against any one of theseclonal populations, because of the small number of each type. Evenmounting a response against one serotype would not protect againstrapidly evolving, new serotypes. The fact that B. burgdorferi hasevolved such an elaborate mechanism for varying the sequence of VlsEindicates the importance of the protein in pathogenesis and/or immuneevasion.

The present invention discloses a repetitive DNA sequence ˜500 bp inlength which is present in multiple, nonidentical copies in a 28 kblinear plasmid of infectious Borrelia burgdorferi, the causative agentof Lyme disease. These DNA sequences encode polypeptides which havesequence similarity to the Variable Major Proteins (VMPs) of relapsingfever Borreliae (such as B. hermsii). VMPs are highly antigenic surfaceproteins which the relapsing fever Borreliae are able to change througha genetic recombination mechanism, thereby evading the immune response.Antibodies against a particular VMP are protective, resulting in rapidclearance of bacteria of the corresponding serotype. In B. burgdorferi,VMP-like sequences (vls) are present on a 28 kb linear plasmid, and thisplasmid appears to encode virulence factor(s) required for infectivity.The sequence of a 16 kb region of this plasmid contains at least 20copies of the VMP-like sequence.

The inventors have identified genes and gene products that appear to beimportant in the infectivity and pathogenesis of B. burgdorferi. Inprevious studies (Norris et al., 1995), it was shown that clonalpopulations of B. burgdorferi isolated after 5 to 15 in vitro passagesvaried significantly in their infectivity in the C3H/HeN mouse model.So-called high-infectivity and low-infectivity clones differed by500-fold in their median infectious dose (1.8×10² vs. 1×10⁵), yetexhibited no obvious differences in terms of protein content (asdetermined by two dimensional gel electrophoresis and silver staining)or plasmid content (determined by agarose gel electrophoresis andethidium bromide staining). However, by using subtractive hybridizationbetween DNA of high- and low-infectivity organisms, specific sequencesthat differed between the two types have been identified. Thesesequences have been characterized as VMP-like sequences (vls), nowidentified for the first time in B. burgdorferi.

In initial studies, high-passage (HP) and low-passage (LP) unclonedpopulations of B. burgdorferi strain B31 were used as a source of DNAfor subtractive hybridization. HP B31 was cultured in vitro for ˜1,000passages and found to be noninfectious, whereas LP B31passages in vitro˜5 times remains infectious in the C3H/HeN mouse model. The plasmid DNAof each strain was purified. The DNA of HP B31was randomly sheared byultrasonication, whereas the DNA of LP B31 was digested to completionwith the restriction enzyme Sau3AI The DNA of the two strains wasdenatured by heating to 100° C., mixed at a ratio of 50:1 HP DNA to LPDNA, and allowed to hybridize with the sheared HP DNA; as a result, theSau3AI restriction sites were not regenerated. Unique segments of the LPDNA tend to hybridize with the complementary LP DNA strand, and theSau3AI “sticky ends” are regenerated. A portion of the hybridizedmixture was ligated into pBluescript II SK- (Stratagene) that had beentreated previously with BamHI and alkaline phosphatase. The ligatedpreparation was used to transform E. coli XL-1 Blue cells, andtransformants were selected by plating the bacteria on Luria broth (LB)agar plates containing ampicillin and isopropyl thiogalactopyranoside(IPTG) and 5-bromo-4-chloro-3-indolyl-D-galactoside (X-gal). Anyresulting white colonies (E. Coli containing a plasmid with a DNAinsert) were selected for further study and sequence analysis.

One of the resulting recombinant plasmids, designated pJRZ53, containedDNA encoding a single, contiguous open reading frame (ORF) 562 bp inlength. The deduced amino acid sequence of this ORF had significanthomology with Vmp proteins of B. hermsii, most notably Vmp17, Vmp21,Vmp7, and Vmp25 (27.2 to 20.3% identity, 50.0 to 56.8% similarity).Hybridization of pJRZ53 with Southern blots of B. burgdorferi plasmidsshowed that this VMP-like DNA sequence was localized on a 28 kb linearplasmid.

Additional DNA recombinants containing sequences hybridizing with pJRZ53were derived from a PstI library of B. burgdorferi B31 plasmid DNA. B.burgdorferi B31 plasmid DNA was treated with several restriction enzymesto determine the best combination for cloning a larger fragmentcontaining the pJYZ53 sequence. Surprisingly, numerous bands hybridizingwith the pJRZ53 probe were present in DNA digested with PstI, RsaI,Sau3AI and other enzymes. This result demonstrated that multiplesequences resembling the pJRZ53 insert were present in the 28 kbplasmid.

Several additional recombinant clones were obtained by treating B.burgdorferi plasmid DNA with PstI, ligating the fragments intopBluescript II SK-, transforming E. coli with the resultingrecombinants, and screening the library for hybridization with thepHRZ53. Sequence determinations of these recombinant clones confirmedthe presence of multiple sequences that were highly homologous butnonidentical to the pHRZ53 sequence. This homology at the DNA andprotein levels is exemplified by the comparison of the two contiguousrepeats found in pHRZ53-31, an independently derived 1143 bp PstI clonethat overlaps the pJRZ53 sequence. Alignment of the 5′ and 3′ regions ofpJRZ53-31 shows highly homologous repeats in the DNA sequence ofrecombinant clone pJRZ53-31. The DNA sequence from the 5′ (nt 1-578) and3′ (nt 579-1143) regions were aligned using the GCG program GAP. Thereis 93% identity between the 5′ and 3′ regions. The deduced amino acidsequences of the DNA regions were aligned using the GCG program GAP. Theoverall sequence similarity and identity are 92% and 85%, respectively.

Subsequent studies used clonal populations of B. burgdorferi obtained bysubsurface colony formation of passage 5 organisms on agar plates(Norris et al., 1995). These clones were characterized in terms ofinfectivity, and were subdivided into high-infectivity andlow-infectivity phenotypes. pJRZ53 hybridized with a 28 kb band in 6/9B31 clones and 7/10 Sh2-2-82 clones. All 12 highly infectious clonescontained the plasmid, whereas 6 of 7 low infectivity clones lacked theplasmid (Table 2). There was correlation of the presence of a 28 kbplasmid containing the pJRZ53 sequence with infectivity of B.burgdorferi clonal populations. Genomic DNA preparations from 10Sh2-2-82 clones and 9 B31 clones (Norris et al., 1995) were subjected topulsed field gel electrophoresis and transferred to nylon membranes. The562 bp pJRZ53 insert labeled with ³²P was hybridized to the Southernblots. Controls consisted of uncloned populations of high-passage,noninfectious B31 (−) and low-passage, infectious B31 (+). All highinfectivity clones (+) possessed a 28 kb plasmid that hybridized withpJRZ53, whereas only 1/7 low-infectivity clones (−) had the plasmid.

Thus there is a strong correlation between the presence of the 28 kbplasmid and infectivity. Plasmid profiles in the same gels used forSouthern blot hybridization did not reveal any difference in ethidiumbromide staining in the region of the 28 kb plasmid, due to the presenceof several other comigrating plasmids. The one low infectivity clonethat contained the plasmid may lack a functional gene or genes encodingother virulence factors.

TABLE 2 Correlation Between Infectivity and the Presence of the 28 kbLinear Plasmid Hybridizing with the pJRZ53 Sequence Number of clonescontaining the 28 Clonal Populations kb plasmid/total B31, highinfectivity 5/5 B31, low infectivity 1/4 Sh2, high infectivity 7/7 Sh2,low infectivity 0/3

A map of the 28 kb linear plasmid (designated pBb28L) showed a 16 kbfragment of pBb28L of the bacterial clone B31-5A3 had been cloned intothe vector lambda Dash II (Stratagene, LaJolla, Calif.). Briefly, apreparation of plasmid DNA was treated with S1 nuclease to disrupt thecovalently closed ends (telomeres) of the linear plasmids. Aftertreatment with Klenow fragment of DNA polymerase, an oligonucleotidelinker containing an EcoRI site was ligated onto the ends. Thepreparation was then treated with EcoRI (to cleave the DNA both at thelinker and at a previously mapped internal EcoRI site) and ligated intothe EcoRI site of lambda Dash II. Clones containing the pJRZ53 sequencewere identified by hybridization, and included two overlapping clones 12kb and 16 kb in length. Partial sequence analysis of the sequence of the16 kb fragment revealed the presence of at least 17 VMP-like sequenceswithin this region.

Over 9,500 bp of DNA sequence from the B. burgdorferi DNA insert inLambda DashII Clone 12-1 have been obtained. This sequence wasdetermined through automated sequence analysis (using T3, T7, andinternal primers) of DashII 12-1 itself, as well as of portions of 12-1cloned into pBluescript using fragments obtained by randomDNasel-mediated cleavage or by digestion with PstI or RsaI. Sequenceswere assembled using the GCG program Gelassemble and have an averageredundancy of ˜4 fold. The high degree of sequence identity amongdifferent regions required careful verification of sequence differencesand manual alignment of sequences in some instances.

The sequences obtained indicate the presence of at least one openreading frame representing an expression site (vlsE1) and at least 16additional nonidentical, apparently ‘silent’ (nonexpressed) vlscassettes. A consensus ribosome binding site (RBS, underlined) islocated 8 nucleotides (nt) upstream of the predicted translational startsite at nt 75-77. The predicted product, VlsE1, has a molecular weightof 35,881 kDa. The first 19 amino acids of the predicted N-terminuscontain a possible signal peptide sequence, with a motif of a chargedN-terminus, a hydrophobic region, and a potential signal peptidase IIcleavage site (FINC, double-underlined) resembling those found in otherBorrelial lipoproteins. The predicted polypeptide size after cleavage atthis site is 33,957 kDa, and the predicted isoelectric point is 7.3.Except for the signal peptide, the predicted protein is largelyhydrophilic. The putative stop codon is located at nt 1143-1145, only 82nt from the telomeric end of pBb28L.

Expression of vlsE1 in the high infectivity B. burgdorferi B31 Clone 3was verified by Northern blot analysis and reverse transcriptasepolymerase chain reaction (RT-PCR™). Hybridization of radiolabelledpJRZ53 insert to blots of RNA separated by agarose electrophoresisindicated the presence of a transcript containing a homologous sequence.For RT-PCR™, primers corresponding to nt 835-857 (plus strand) and nt1010-1032 (minus strand) of the sequence in FIG. 2C were constructed.The minus strand primer was used in combination with AMV reversetranscriptase and RNA isolated from B31 clone 3 to produce a cDNAproduct. The cDNA was then amplified by standard PCR™ using the plus andminus primers, resulting in a 198 bp product detectable by ethidiumbromide staining of agarose gels. The PCR™ product was ligated into thepCRII vector (Invitrogen), and three independently-derived clonesyielded sequences identical to that shown in SEQ ID NO:1 and SEQ IDNO:3. Control preparations consisted of reactions identical except forthe omission of reverse transcriptase; no product was detected. Thisresult demonstrated that vlsE1 is transcribed in B. burgdorferi B31Clone 3 organisms.

The DNA sequence of a proposed ‘storage site’ contains at least 15contiguous copies of the vls sequence of SEQ ID NO:1 and SEQ ID NO:3.The beginning and end of each vls ‘cassette’ was selected to match therepetitive sequence (vls1) in vlsE1. The vls cassettes identified thusfar range from 474 to 582 nt in length; length variation is primarilydue to short insertions or deletions in multiples of three nucleotides,indicating selective preservation of the open reading frames. Longerdeletions are seen in vls7, vls8, and vls10. vls14 and vls16 eachcontain one frameshift, and vls1 contains one stop codon. Otherwise, the7766 bp sequence of SEQ ID NO:1 and SEQ ID NO:3 represents onecontiguous open reading frame.

The vls cassettes exhibit a remarkable degree of sequence conservationat both the DNA and encoded amino acid levels, see Figures. Nucleotidesequences of B. burgdorferi B31 vls were aligned using the GCG programPILEUP. vls1 corresponds to nt 420-1003 in the sequence of FIG. 4 andFIG. 5. When compared to vls1 using GCG program GAP, the vls sequenceshave 90.0% to 96.1% nucleotide sequence identity (FIG. 6), and 76.9% to91.4% predicted amino acid sequence identity (FIG. 7). None of the vlscopies identified thus far have complete sequence identity, but all areclosely related.

Table 3 shows the vls segments identified and indicates the positions atwhich the segments may be found as part of SEQ ID NO:1 and SEQ ID NO:3.Repeat recombinant segments are identified as “repeats”.

TABLE 3 CAS- SETTE POSITION IN REPEAT position in (vls) SEQ ID NO: 3 SEQID NO: 3 vls 2 <205>-711  711-727 (truncated at 5′ end) vls 3  712-12931293-1309 vls 4 1294-1869 1869-1885 vls 5 1870-2439 2439-2456 vls 62440-3009 3009-3025 vls 7 3010-3483 3483-3499 vls 8 3484-3990 3990-4006vls 9 3991-4548 4548-4557 vls 10 4549-5058 5058-5074 vls 11 5059-56525652-5668 vls 12 4653-6219 6219-6253 vls 13 6220-6789 6789-6805 vls 146846-7373 7373-7389 vls 15 7274-7946 7946-7962 vls 16 7947-8000

The degree of sequence similarity between the VMP-like sequences and B.hermsii VMP proteins were exemplified by an alignment of the predictedtranslation product of vlsE1 with some of the most similar VMP sequences(vmp 17, vmp 21, vmp7). Regions of similarity are interspersed amongareas of low sequence identity. The G+C contents of the B. burgdorferiVMP-like sequences are quite high (e.g., 49.9% for pJRZ53-31) ascompared to the genomic B. burgdorferi G+C content (27 to 30%) or thatof B. hermsii VMP genes (e.g., 37% for vmp17). The sequence similarityat the protein level may be due to divergent or convergent evolution. Itis also possible that the VMP-like sequences were acquired from anotherorganism, given the different G+C content.

Alignment of either the DNA sequences or the deduced amino acidsequences of the open reading frames reveals the presence of bothconserved and variable regions of the repetitive sequence. The conservedsequences may represent ‘framework’ regions important in the overallstructure of the polypeptides, whereas the variable sequences mayproduce different epitopes. It is contemplated that protectiveantibodies can be produced against either the conserved or variableportions of the putative amino acid sequences.

The expressed copy of vls (vlsE1) has been identified and sequenced. Asegment of the vlsE gene corresponding to the cassette region has beensubcloned into the pGEX-2T expression vector, and the resulting GST-vls1fusion protein product produced and purified. Antibodies against therecombinant protein have been used for identification of the nativeprotein in SDS-PAGE and two dimensional gel electrophoresis patterns ofB. burgdorferi proteins by immunoblotting. Infected patients and animalsproduced antibodies against the protein which were detected byimmunoblot analysis using the recombinant protein as antigen (FIGS. 6C,6D and 6E). In addition, the purified recombinant protein may be usedfor immunization of mice and other animals to determine whetherantibodies or cellular responses against the protein are protectiveagainst infection with B. burgdorferi and other Lyme disease Borreliae.Such animal studies would determine the feasibility of vaccination ofhumans and animals with Vls protein sequences or DNA sequences forimmunoprophylaxis.

ELISAs

ELISAs may be used in conjunction with the invention. In an ELISA assay,proteins or peptides incorporating Borrelia VMP-like antigenic sequencesare immobilized onto a selected surface, preferably a surface exhibitinga protein affinity such as the wells of a polystyrene microtiter plate.After washing to remove incompletely adsorbed material, it is desirableto bind or coat the assay plate wells with a nonspecific protein that isknown to be antigenically neutral with regard to the test antisera suchas bovine serum albumin (BSA), casein or solutions of powdered milk.This allows for blocking of nonspecific adsorption sites on theimmobilizing surface and thus reduces the background caused bynonspecific binding of antisera onto the surface.

After binding of antigenic material to the well, coating with anon-reactive material to reduce background, and washing to removeunbound material, the immobilizing surface is contacted with theantisera or clinical or biological extract to be tested in a mannerconducive to immune complex (antigen/antibody) formation. Suchconditions preferably include diluting the antisera with diluents suchas BSA, bovine gamma globulin (BGG) and phosphate buffered saline(PBS)/Tween®. These added agents also tend to assist in the reduction ofnonspecific background. The layered antisera is then allowed to incubatefor from about 2 to about 4 hr, at temperatures preferably on the order25° to about 27° C. Following incubation, the antisera-contacted surfaceis washed so as to remove non-immunocomplexed material. A preferredwashing procedure includes washing with a solution such as PBS/Tween®,or borate buffer.

Following formation of specific immunocomplexes between the test sampleand the bound antigen, and subsequent washing, the occurrence and evenamount of immunocomplex formation may be determined by subjecting sameto a second antibody having specificity for the first. To provide adetecting means, the second antibody will preferably have an associatedenzyme that will generate a color development upon incubating with anappropriate chromogenic substrate. Thus, for example, one will desire tocontact and incubate the antisera-bound surface with a urease, alkalinephosphatase or peroxidase-conjugated anti-human IgG for a period of timeand under conditions which favor the development of immunocomplexformation (e.g. incubation for 2 hr at room temperature in aPBS-containing solution such as PBS/Tween®.

After incubation with the second enzyme-tagged antibody, and subsequentto washing to remove unbound material, the amount of label is quantifiedby incubation with a chromogenic substrate such as urea and bromocresolpurple or 2,2′-azino-di-(3-ethyl-benzthiazoline)-6-sulfonic acid (ABTS)and H₂O₂, in the case of peroxidase as the enzyme label. Quantitation isthen achieved by measuring the degree of color generation, e.g., using avisible spectrum spectrophotometer.

Epitopic Core Sequences

The present invention is also directed to protein or peptidecompositions. free from total cells and other peptides, which comprise apurified protein or peptide which incorporates an epitope that isimmunologically cross-reactive with one or more anti-Borrelia VMP-likeantibodies.

As used herein, the term “incorporating an epitope(s) that isimmunologically cross-reactive with one or more anti-VMP-likeantibodies” is intended to refer to a peptide or protein antigen whichincludes a primary, secondary or tertiary structure similar to anepitope located within a Borrelia VMP-like polypeptide. The level ofsimilarity will generally be to such a degree that monoclonal orpolyclonal antibodies directed against the Borrelia VMP-like polypeptidewill also bind to, react with, or otherwise recognize, thecross-reactive peptide or protein antigen. Various immunoassay methodsmay be employed in conjunction with such antibodies, such as, forexample, Western blotting, ELISA, RIA, and the like, all of which areknown to those of skill in the art.

The identification of Borrelia VMP-like epitopes, and/or theirfunctional equivalents, suitable for use in vaccines is a relativelystraightforward matter. For example, one may employ the methods of Hopp,as taught in U.S. Pat. No. 4,554,101, incorporated herein by reference,which teaches the identification and preparation of epitopes from aminoacid sequences on the basis of hydrophilicity. The methods described inseveral other papers, and software programs based thereon, can also beused to identify epitopic core sequences (see, for example, Jameson andWolf, 1988; Wolf et al., 1988; U.S. Pat. No. 4,554,101). The amino acidsequence of these “epitopic core sequences” may then be readilyincorporated into peptides, either through the application of peptidesynthesis or recombinant technology.

Preferred peptides for use in accordance with the present invention willgenerally be on the order of about 5 to about 25 amino acids in length,and more preferably about 8 to about 20 amino acids in length. It isproposed that shorter antigenic Borrelia VMP-like-derived peptidesequences will provide advantages in certain circumstances, for example,in the preparation of vaccines or in immunologic detection assays.Exemplary advantages include the ease of preparation and purification,the relatively low cost and improved reproducibility of production, andadvantageous biodistribution.

It is proposed that particular advantages of the present invention maybe realized through the preparation of synthetic peptides which includemodified and/or extended epitopic/immunogenic core sequences whichresult in a “universal” epitopic peptide directed to Borrelia VMP-likeand Borrelia VMP-like-related sequences. It is proposed that theseregions represent those which are most likely to promote T-cell orB-cell stimulation in an animal, and, hence, elicit specific antibodyproduction in such an animal.

An epitopic core sequence, as used herein, is a relatively short stretchof amino acids that is “complementary” to, and therefore will bind,antigen binding sites on transferring-binding protein antibodies.Additionally or alternatively, an epitopic core sequence is one thatwill elicit antibodies that are cross-reactive with antibodies directedagainst the peptide compositions of the present invention. It will beunderstood that in the context of the present disclosure, the term“complementary” refers to amino acids or peptides that exhibit anattractive force towards each other. Thus, certain epitope coresequences of the present invention may be operationally defined in termsof their ability to compete with or perhaps displace the binding of thedesired protein antigen with the corresponding protein-directedantisera.

In general, the size of the polypeptide antigen is not believed to beparticularly crucial, so long as it is at least large enough to carrythe identified core sequence or sequences. The smallest useful coresequence expected by the present disclosure would generally be on theorder of about 5 amino acids in length, with sequences on the order of 8or 25 being more preferred. Thus, this size will generally correspond tothe smallest peptide antigens prepared in accordance with the invention.However, the size of the antigen may be larger where desired, so long asit contains a basic epitopic core sequence.

The identification of epitopic core sequences is known to those of skillin the art, for example, as described in U.S. Pat. No. 4,554,101,incorporated herein by reference, which teaches the identification andpreparation of epitopes from amino acid sequences on the basis ofhydrophilicity. Moreover, numerous computer programs are available foruse in predicting antigenic portions of proteins (see e.g., Jameson andWolf, 1988; Wolf et al., 1988). Computerized peptide sequence analysisprograms (e.g., DNAStar® software, DNAStar, Inc., Madison. Wis.) mayalso be useful in designing synthetic Borrelia VMP-like peptides andpeptide analogs in accordance with the present disclosure.

Syntheses of epitopic sequences or peptides which include an antigenicepitope within their sequence, are readily achieved using conventionalsynthetic techniques such as the solid phase method (e.g., through theuse of commercially available peptide synthesizer such as an AppliedBiosystems Model 430A Peptide Synthesizer). Peptide antigens synthesizedin this manner may then be aliquoted in predetermined amounts and storedin conventional manners, such as in aqueous solutions or, even morepreferably, in a powder or lyophilized state pending use.

In general, due to the relative stability of peptides, they may bereadily stored in aqueous solutions for fairly long periods of time ifdesired, e.g., up to six months or more, in virtually any aqueoussolution without appreciable degradation or loss of antigenic activity.However, where extended aqueous storage is contemplated it willgenerally be desirable to include agents including buffers such as Trisor phosphate buffers to maintain a pH of about 7.0 to about 7.5.Moreover, it may be desirable to include agents which will inhibitmicrobial growth, such as sodium azide or Merthiolate. For extendedstorage in an aqueous state it will be desirable to store the solutionsat 4° C., or more preferably, frozen. Of course, where the peptides arestored in a lyophilized or powdered state, they may be stored virtuallyindefinitely, e.g., in metered aliquots that may be rehydrated with apredetermined amount of water (preferably distilled) or buffer prior touse.

Immunoprecipitation

The antibodies of the present invention are particularly useful for theisolation of antigens by immunoprecipitation. Immunoprecipitationinvolves the separation of the target antigen component from a complexmixture, and is used to discriminate or isolate minute amounts ofprotein. For the isolation of membrane proteins cells must besolubilized into detergent micelles. Nonionic salts are preferred, sinceother agents such as bile salts, precipitate at acid pH or in thepresence of bivalent cations.

In an alternative embodiment the antibodies of the present invention areuseful for the close juxtaposition of two antigens. This is particularlyuseful for increasing the localized concentration of antigens, e.g.,enzyme-substrate pairs.

Western Blots

The compositions of the present invention will find great use inimmunoblot or western blot analysis. The anti-Borrelia VMP-likeantibodies may be used as high-affinity primary reagents for theidentification of proteins immobilized onto a solid support matrix, suchas nitrocellulose, nylon or combinations thereof. In conjunction withimmunoprecipitation, followed by gel electrophoresis, these may be usedas a single step reagent for use in detecting antigens against whichsecondary reagents used in the detection of the antigen cause an adversebackground. This is especially useful when the antigens studied areimmunoglobulins (precluding the use of immunoglobulins binding bacterialcell wall components), the antigens studied cross-react with thedetecting agent, or they migrate at the same relative molecular weightas a cross-reacting signal.

Immunologically-based detection methods for use in conjunction withWestern blotting include enzymatically-, radiolabel-, orfluorescently-tagged secondary antibodies against the toxin moiety areconsidered to be of particular use in this regard.

Vaccines

The present invention contemplates vaccines for use in both active andpassive immunization embodiments. Immunogenic compositions, proposed tobe suitable for use as a vaccine may be prepared most readily directlyfrom immunogenic Borrelia VMP-like peptides prepared in a mannerdisclosed herein. Preferably the antigenic material is extensivelydialyzed to remove undesired small molecular weight molecules and/orlyophilized for more ready formulation into a desired vehicle.

The preparation of vaccines which contain Borrelia VMP-like peptidesequences as active ingredients is generally well understood in the art,as exemplified by U.S. Pat. Nos. 4,608,251; 4,601,903; 4,599,231;4,599,230; 4,596,792; and 4,578,770, all incorporated herein byreference. Typically, such vaccines are prepared as injectables. Eitheras liquid solutions or suspensions: solid forms suitable for solutionin, or suspension in, liquid prior to injection may also be prepared.The preparation may also be emulsified. The active immunogenicingredient is often mixed with excipients which are pharmaceuticallyacceptable and compatible with the active ingredient. Suitableexcipients are, for example, water, saline, dextrose, glycerol, ethanol,or the like and combinations thereof. In addition, if desired, thevaccine may contain minor amounts of auxiliary substances such aswetting or emulsifying agents, pH buffering agents, or adjuvants whichenhance the effectiveness of the vaccines.

Vaccines may be conventionally administered parenterally, by injection,for example, either subcutaneously or intramuscularly. Additionalformulations which are suitable for other modes of administrationinclude suppositories and, in some cases, oral formulations. Forsuppositories, traditional binders and carriers may include, forexample, polyalkalene glycols or triglycerides: such suppositories maybe formed from mixtures containing the active ingredient in the range ofabout 0.5% to about 10%, preferably about 1 to about 2%. Oralformulations include such normally employed excipients as, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate and the like. Thesecompositions take the form of solutions, suspensions, tablets, pills,capsules, sustained release formulations or powders and contain about 10to about 95% of active ingredient, preferably about 25 to about 70%.

The Borrelia VMP-like-derived peptides of the present invention may beformulated into the vaccine as neutral or salt forms.Pharmaceutically-acceptable salts. include the acid addition salts(formed with the free amino groups of the peptide) and those which areformed with inorganic acids such asp for example, hydrochloric orphosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups mayalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, 2-ethylamino ethanol,histidine, procaine, and the like.

The vaccines are administered in a manner compatible with the dosageformulation, and in such amount as will be therapeutically effective andimmunogenic. The quantity to be administered depends on the subject tobe treated, including, e.g., the capacity of the individual's immunesystem to synthesize antibodies, and the degree of protection desired.Precise amounts of active ingredient required to be administered dependon the judgment of the practitioner. However, suitable dosage ranges areof the order of several hundred micrograms active ingredient pervaccination. Suitable regimes for initial administration and boostershots are also variable, but are typified by an initial administrationfollowed by subsequent inoculations or other administrations.

The manner of application may be varied widely. Any of the conventionalmethods for administration of a vaccine are applicable. These arebelieved to include oral application on a solid physiologicallyacceptable base or in a physiologically acceptable dispersion,parenterally, by injection or the like. The dosage of the vaccine willdepend on the route of administration and will vary according to thesize of the host.

Various methods of achieving adjuvant effect for the vaccine includesuse of agents such as aluminum hydroxide or phosphate (alum), commonlyused as about 0.05 to about 0.1% solution in phosphate buffered saline,admixture with synthetic polymers of sugars (Carbopol®) used as an about0.25% solution, aggregation of the protein in the vaccine by heattreatment with temperatures ranging between about 70° to about 101° C.for a 30-second to 2-minute period, respectively. Aggregation byreactivating with pepsin treated (Fab) antibodies to albumin, mixturewith bacterial cells such as C. parvum or endotoxins orlipopolysaccharide components of Gram-negative bacteria, emulsion inphysiologically acceptable oil vehicles such as mannide mono-oleate(Aracel A) or emulsion with a 20% solution of a perfluorocarbon(Fluosol-DA®) used as a block substitute may also be employed.

In many instances, it will be desirable to have multiple administrationsof the vaccine, usually not exceeding six vaccinations, more usually notexceeding four vaccinations and preferably one or more, usually at leastabout three vaccinations. The vaccinations will normally be at from twoto twelve week intervals, more usually from three to five weekintervals. Periodic boosters at intervals of 1-5 years, usually threeyears, will be desirable to maintain protective levels of theantibodies. The course of the immunization may be followed by assays forantibodies for the supernatant antigens. The assays may be performed bylabeling with conventional labels, such as radionuclides, enzymes,fluorescents, and the like. These techniques are well known and may befound in a wide variety of patents, such as U.S. Pat. Nos. 3,791,932;4,174,384 and 3,949,064, as illustrative of these types of assays.

DNA Segments

In other embodiments, it is contemplated that certain advantages will begained by positioning the coding DNA segment under the control of arecombinant, or heterologous, promoter. As used herein, a recombinant orheterologous promoter is intended to refer to a promoter that is notnormally associated with a DNA segment encoding a Borrelia VMP-likepeptide in its natural environment. Such promoters may include promotersnormally associated with other genes, and/or promoters isolated from anyviral, prokaryotic (e.g., bacterial), eukaryotic (e.g., fungal, yeast,plant, or animal) cell, and particularly those of mammalian cells.Naturally, it will be important to employ a promoter that effectivelydirects the expression of the DNA segment in the cell type, organism, oreven animal, chosen for expression. The use of promoter and cell typecombinations for protein expression is generally known to those of skillin the art of molecular biology, for example, see Sambrook et al., 1989.The promoters employed may be constitutive, or inducible, and can beused under the appropriate conditions to direct high level expression ofthe introduced DNA segment, such as is advantageous in the large-scaleproduction of recombinant proteins or peptides. Appropriatepromoter/expression systems contemplated for use in high-levelexpression include, but are not limited to, the Pichia expression vectorsystem (Pharmacia LKB Biotechnology), a baculovirus system forexpression in insect cells, or any suitable yeast or bacterialexpression system.

In connection with expression embodiments to prepare recombinantproteins and peptides, it is contemplated that longer DNA segments willmost often be used, with DNA segments encoding the entire peptidesequence being most preferred. However, it will be appreciated that theuse of shorter DNA segments to direct the expression of BorreliaVMP-like peptides or epitopic core regions, such as may be used togenerate anti-Borrelia VMP-like antibodies, also falls within the scopeof the invention. DNA segments that encode Borrelia VMP-like peptideantigens from about 10 to about 100 amino acids in length, or morepreferably, from about 20 to about 80 amino acids in length, or evenmore preferably, from about 30 to about 70 amino acids in length arecontemplated to be particularly useful.

In addition to their use in directing the expression of BorreliaVMP-like peptides of the present invention, the nucleic acid sequencescontemplated herein also have a variety of other uses. For example, theyalso have utility as probes or primers in nucleic acid hybridizationembodiments. As such, it is contemplated that nucleic acid segments thatcomprise a sequence region that consists of at least an about14-nucleotide long contiguous sequence that has the same sequence as, oris complementary to, an about 14-nucleotide long contiguous DNA segmentof SEQ ID NO:1 and SEQ ID NO:3 will find particular utility. Longercontiguous identical or complementary sequences, e.g., those of about20, 30, 40, 50, 100, 200, (including all intermediate lengths) and eventhose up to and including about 1227-bp (full-length) sequences willalso be of use in certain embodiments.

The ability of such nucleic acid probes to specifically hybridize toBorrelia/a VMP-like-encoding sequences will enable them to be of use indetecting the presence of complementary sequences in a given sample.However, other uses are envisioned, including the use of the sequenceinformation for the preparation of mutant species primers, or primersfor use in preparing other genetic constructions.

Nucleic acid molecules having sequence regions consisting of contiguousnucleotide stretches of about 14, 15-20, 30, 40, 50, or even of about100 to about 200 nucleotides or so, identical or complementary to theDNA sequence of SEQ ID NO:1 and SEQ ID NO:3, are particularlycontemplated as hybridization probes for use in, e.g., Southern andNorthern blotting. Smaller fragments will generally find use inhybridization embodiments, wherein the length of the contiguouscomplementary region may be varied, such as between about 10-14 and tipto about 100 nucleotides, but larger contiguous complementary stretchesmay be used, according to the length complementary sequences one wishesto detect.

The use of a hybridization probe of about 14 nucleotides in lengthallows the formation of a duplex molecule that is both stable andselective. Molecules having contiguous complementary sequences overstretches greater than 14 bases in length are generally preferred,though, in order to increase stability and selectivity of the hybrid,and thereby improve the quality and degree of specific hybrid moleculesobtained. One will Generally prefer to design nucleic acid moleculeshaving gene-complementary stretches of about 15 to about 20 contiguousnucleotides, or even longer where desired.

Of course. fragments may also be obtained by other techniques such as,e.g., by mechanical shearing or by restriction enzyme digestion. Smallnucleic acid segments or fragments may be readily prepared by, forexample, directly synthesizing the fragment by chemical means, as iscommonly practiced using an automated oligonucleotide synthesizer. Also,fragments may be obtained by application of nucleic acid reproductiontechnology, such as PCR™, by introducing selected sequences intorecombinant vectors for recombinant production, and by other recombinantDNA techniques generally known to those of skill in the art of molecularbiology.

Accordingly, the nucleotide sequences of the invention may be used fortheir ability to selectively form duplex molecules with complementarystretches of DNA fragments. Depending on the application envisioned, onewill desire to employ varying conditions of hybridization to achievevarying degrees of selectivity of probe towards target sequence. Forapplications requiring high selectivity, one will typically desire toemploy relatively stringent conditions to form the hybrids, e.g.,conditions of high stringency where one will select relatively low saltand/or high temperature conditions, such as provided by about 0.02 M toabout 0.15 M NaCl at temperatures of about 50° C. to about 70° C. Suchselective conditions tolerate little, if any, mismatch between the probeand the template or target strand, and would be particularly suitablefor isolating Borrelia VMP-like-encoding DNA segments. Detection of DNAsegments via hybridization is well-known to those of skill in the art,and the teachings of U.S. Pat. Nos. 4,965,188 and 5,176,995 (eachincorporated herein by reference) are exemplary of the methods ofhybridization analyses. Teachings such as those found in the texts ofMaloy et al., 1994; Segal, 1976; Prokop, 1991; and Kuby, 1994, areparticularly relevant.

Of course, for some applications, for example, where one desires toprepare mutants employing a mutant primer strand hybridized to anunderlying template or where one seeks to isolate BorreliaVMP-like-encoding sequences from related species, functionalequivalents, or the like, less stringent hybridization conditions willtypically be needed in order to allow formation of the heteroduplex. Inthese circumstances, one may desire to employ conditions such as about0.15 M to about 0.9 M salt, at temperatures ranging from about 20° C. toabout 55° C. Cross-hybridizing species can thereby be readily identifiedas positively hybridizing signals with respect to controlhybridizations. In any case, it is generally appreciated that conditionscan be rendered more stringent by the addition of increasing amounts offormamide, which serves to destabilize the hybrid duplex in the samemanner as increased temperature. Thus, hybridization conditions can bereadily manipulated, and thus will generally be a method of choicedepending on the desired results.

In certain embodiments, it will be advantageous to employ nucleic acidsequences of the present invention in combination with an appropriatemeans, such as a label, for determining hybridization. A wide variety ofappropriate indicator means are known in the art, including fluorescent,radioactive, enzymatic or other ligands, such as avidin/biotin, whichare capable of giving a detectable signal. In preferred embodiments, onewill likely desire to employ a fluorescent label or an enzyme tag, suchas urease, alkaline phosphatase or peroxidase, instead of radioactive orother environmental undesirable reagents. In the case of enzyme tags,colorimetric indicator substrates are known that can be employed toprovide a means visible to the human eye or spectrophotometrically, toidentify specific hybridization with complementary nucleicacid-containing samples.

In general, it is envisioned that the hybridization probes describedherein will be useful both as reagents in solution hybridization as wellas in embodiments employing a solid phase. In embodiments involving asolid phase, the test DNA (or RNA) is adsorbed or otherwise affixed to aselected matrix or surface. This fixed, single-stranded nucleic acid isthen subjected to specific hybridization with selected probes underdesired conditions. The selected conditions will depend on theparticular circumstances based on the particular criteria required(depending, for example, on the G+C content, type of target nucleicacid, source of nucleic acid, size of hybridization probe, etc.).Following washing of the hybridized surface so as to removenonspecifically bound probe molecules, specific hybridization isdetected, or even quantitated, by means of the label.

Biological Functional Equivalents

Modification and changes may be made in the structure of the peptides ofthe present invention and DNA segments which encode them and stillobtain a functional molecule that encodes a protein or peptide withdesirable characteristics. The following is a discussion based uponchanging the amino acids of a protein to create an equivalent, or evenan improved, second-generation molecule. The amino acid changes may beachieved by changing the codons of the DNA sequence, according to thefollowing codon table:

TABLE 4 Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys CUGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu E GAA GAGPhenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine HisH CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Leucine LeuL UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAUProline Pro P CCA CCC CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGAAGG CGA CGC CGG CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr TACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGGTyrosine Tyr Y UAC UAU

For example, certain amino acids may be substituted for other aminoacids in a protein structure without appreciable loss of interactivebinding capacity with structures such as, for example, antigen-bindingregions of antibodies or binding sites on substrate molecules. Since itis the interactive capacity and nature of a protein that defines thatprotein's biological functional activity, certain amino acid sequencesubstitutions can be made in a protein sequence, and, of course, itsunderlying DNA coding sequence, and nevertheless obtain a protein withlike properties. It is thus contemplated by the inventors that variouschanges may be made in the peptide sequences of the disclosedcompositions, or corresponding DNA sequences which encode said peptideswithout appreciable loss of their biological utility or activity.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982, incorporate herein byreference). It is accepted that the relative hydropathic character ofthe amino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like.

Each amino acid has been assigned a hydropathic index on the basis oftheir hydrophobicity and charge characteristics (Kyte and Doolittle,1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9);alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8);tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2);glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5);lysine (−3.9); and arginine (−4.5).

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e., still obtaina biological functionally equivalent protein. In making such changes,the substitution of amino acids whose hydropathic indices are within ±2is preferred, those which are within ±1 are particularly preferred, andthose within ±0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with a biologicalproperty of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).

It is understood that an amino acid can be substituted for anotherhaving a similar hydrophilicity value and still obtain a biologicallyequivalent, and in particular, an immunologically equivalent protein. Insuch changes, the substitution of amino acids whose hydrophilicityvalues are within ±2 is preferred, those which are within ±1areparticularly preferred, and those within ±0.5 are even more particularlypreferred.

As outlined above, amino acid substitutions are generally thereforebased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions which take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

Site-specific Mutagenesis

Site-specific mutagenesis is a technique useful in the preparation ofindividual peptides, or biologically functional equivalent proteins orpeptides, through specific mutagenesis of the underlying DNA. Thetechnique further provides a ready ability to prepare and test sequencevariants, for example, incorporating one or more of the foregoingconsiderations, by introducing one or more nucleotide sequence changesinto the DNA. Site-specific mutagenesis allows the production of mutantsthrough the use of specific oligonucleotide sequences which encode theDNA sequence of the desired mutation, as well as a sufficient number ofadjacent nucleotides, to provide a primer sequence of sufficient sizeand sequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Typically, a primer of about 17 to 25nucleotides in length is preferred, with about 1 to 10 residues on bothsides of the junction of the sequence being altered.

In general, the technique of site-specific mutagenesis is well known inthe art, as exemplified by various publications. As will be appreciated,the technique typically employs a phage vector which exists in both asingle stranded and double stranded form. Typical vectors useful insite-directed mutagenesis include vectors such as the M13 phage. Thesephage are readily commercially available and their use is generally wellknown to those skilled in the art. Double stranded plasmids are alsoroutinely employed in site directed mutagenesis which eliminates thestep of transferring the gene of interest from a plasmid to a phage.

In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector or melting apartof two strands of a double stranded vector which includes within itssequence a DNA sequence which encodes the desired peptide. Anoligonucleotide primer bearing the desired mutated sequence is prepared,generally synthetically. This primer is then annealed with thesingle-stranded vector, and subjected to DNA polymerizing enzymes suchas E. coli polymerase I Klenow fragment, in order to complete thesynthesis of the mutation-bearing strand. Thus, a heteroduplex is formedwherein one strand encodes the original non-mutated sequence and thesecond strand bears the desired mutation. This heteroduplex vector isthen used to transform appropriate cells, such as E. coli cells, andclones are selected which include recombinant vectors bearing themutated sequence arrangement.

The preparation of sequence variants of the selected peptide-encodingDNA segments using site-directed mutagenesis is provided as a means ofproducing potentially useful species and is not meant to be limiting asthere are other ways in which sequence variants of peptides and the DNAsequences encoding them may be obtained. For example, recombinantvectors encoding the desired peptide sequence may be treated withmutagenic agents, such as hydroxylamine, to obtain sequence variants.

Monoclonal Antibodies

Means for preparing and characterizing antibodies are well known in theart (See, e.g., Harlow and Lane, 1988; incorporated herein byreference).

The methods for generating monoclonal antibodies (mAbs) generally beginalong the same lines as those for preparing polyclonal antibodies.Briefly, a polyclonal antibody is prepared by immunizing an animal withan immunogenic composition in accordance with the present invention andcollecting antisera from that immunized animal. A wide range of animalspecies can be used for the production of antisera. Typically the animalused for production of anti-antisera is a rabbit, a mouse, a rat, ahamster, a guinea pig or a goat. Because of the relatively large bloodvolume of rabbits, a rabbit is a preferred choice for production ofpolyclonal antibodies.

As is well known in the art, a given composition may vary in itsimmunogenicity. It is often necessary therefore to boost the host immunesystem, as may be achieved by coupling a peptide or polypeptideimmunogen to a carrier. Exemplary and preferred carriers are keyholelimpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albuminssuch as ovalbumin, mouse serum albumin or rabbit serum albumin can alsobe used as carriers. Means for conjugating a polypeptide to a carrierprotein are well known in the art and include glutaraldehyde,m-maleimidobenzoyl-N-hydroxysuccinimide ester, carbodiimide andbis-biazotized benzidine.

As is also well known in the art, the immunogenicity of a particularimmunogen composition can be enhanced by the use of non-specificstimulators of the immune response, known as adjuvants. Exemplary andpreferred adjuvants include complete Freund's adjuvant (a non-specificstimulator of the immune response containing killed Mycobacteriumtuberculosis), incomplete Freund's adjuvants and aluminum hydroxideadjuvant.

The amount of immunogen composition used in the production of polyclonalantibodies varies upon the nature of the immunogen as well as the animalused for immunization. A variety of routes can be used to administer theimmunogen (subcutaneous, intramuscular, intradermal, intravenous andintraperitoneal). The production of polyclonal antibodies may bemonitored by sampling blood of the immunized animal at various pointsfollowing immunization. A second, booster, injection may also be given.The process of boosting and titering is repeated until a suitable titeris achieved. When a desired level of immunogenicity is obtained, theimmunized animal can be bled and the serum isolated and stored, and/orthe animal can be used to generate mAbs.

mAbs may be readily prepared through use of well-known techniques, suchas those exemplified in U.S. Pat. No. 4,196,265, incorporated herein byreference. Typically, this technique involves immunizing a suitableanimal with a selected immunogen composition. e.g., a purified orpartially purified LCRF protein, polypeptide or peptide. The immunizingcomposition is administered in a manner effective to stimulate antibodyproducing cells. Rodents such as mice and rats are preferred animals,however, the use of rabbit, sheep frog cells is also possible. The useof rats may provide certain advantages (Goding, 1986), but mice arepreferred, with the BALB/c mouse being most preferred as this is mostroutinely used and generally gives a higher percentage of stablefusions.

Following immunization, somatic cells with the potential for producingantibodies, specifically B-lymphocytes (B-cells), are selected for usein the mAb generating protocol. These cells may be obtained frombiopsied spleens, tonsils or lymph nodes, or from a peripheral bloodsample. Spleen cells and peripheral blood cells are preferred, theformer because they are a rich source of antibody-producing cells thatare in the dividing plasmablast stage, and the latter because peripheralblood is easily accessible. Often, a panel of animals will have beenimmunized and the spleen of animal with the highest antibody titer willbe removed and the spleen lymphocytes obtained by homogenizing thespleen with a syringe. Typically, a spleen from an immunized mousecontains approximately 5×10⁷ to 2×10⁸ lymphocytes.

The antibody-producing B lymphocytes from the immunized animal are thenfused with cells of an immortal myeloma cell, generally one of the samespecies as the animal that was immunized. Myeloma cell lines suited foruse in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render then incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas).

Any one of a number of myeloma cells may be used, as are known to thoseof skill in the art (Goding, 1986; Campbell, 1984). For example, wherethe immunized animal is a mouse, one may use P3-X63/Ag8, X63-Ag8.653,NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 andS194/5XX0 Bul; for rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all usefulin connection with human cell fusions.

One preferred murine myeloma cell is the NS-1 myeloma cell line (alsotermed P3-NS-1-Ag4-1), which is readily available from the NIGMS HumanGenetic Mutant Cell Repository by requesting cell line repository numberGM3573. Another mouse myeloma cell line that may be used is the8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cellline.

Methods for generating hybrids of antibody-producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in a 2:1 ratio, though the ratio may vary from about 20:1to about 1:1, respectively, in the presence of an agent or agents(chemical or electrical) that promote the fusion of cell membranes.Fusion methods using Sendai virus have been described (Kohler andMilstein, 1975; 1976), and those using polyethylene glycol (PEG), suchas 37% (v/v) PEG, by Gefter et al., (1977). The use of electricallyinduced fusion methods is also appropriate (Goding, 1986).

Fusion procedures usually produce viable hybrids at low frequencies,about 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose a problem, as theviable, fused hybrids are differentiated from the parental, unfusedcells (particularly the unfused myeloma cells that would normallycontinue to divide indefinitely) by culturing in a selective medium. Theselective medium is generally one that contains an agent that blocks thede novo synthesis of nucleotides in the tissue culture media. Exemplaryand preferred agents are aminopterin, methotrexate, and azaserine.Aminopterin and methotrexate block de novo synthesis of both purines andpyrimidines, whereas azaserine blocks only purine synthesis. Whereaminopterin or methotrexate is used, the media is supplemented withhypoxanthine and thymidine as a source of nucleotides (HAT medium).Where azaserine is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of operatingnucleotide salvage pathways are able to survive in HAT medium. Themyeloma cells are defective in key enzymes of the salvage pathway, e.g.,hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.The B-cells can operate this pathway, but they have a limited life spanin culture and generally die within about two weeks. Therefore, the onlycells that can survive in the selective media are those hybrids formedfrom myeloma and B-cells.

This culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants (after about twoto three weeks) for the desired reactivity. The assay should besensitive, simple and rapid, such as radioimmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays, dot immunobindingassays, and the like.

The selected hybridomas would then be serially diluted and cloned intoindividual antibody-producing cell lines, which clones can then bepropagated indefinitely to provide mAbs. The cell lines may be exploitedfor mAb production in two basic ways. A sample of the hybridoma can beinjected (often into the peritoneal cavity) into a histocompatibleanimal of the type that was used to provide the somatic and myelomacells for the original fusion. The injected animal develops tumorssecreting the specific monoclonal antibody produced by the fused cellhybrid. The body fluids of the animal, such as serum or ascites fluid,can then be tapped to provide mAbs in high concentration. The individualcell lines could also be cultured in vitro, where the mAbs are naturallysecreted into the culture medium from which they can be readily obtainedin high concentrations. mAbs produced by either means may be furtherpurified, if desired, using filtration, centrifugation and variouschromatographic methods such as HPLC or affinity chromatography.

Pharmaceutical Compositions

The pharmaceutical compositions disclosed herein may be orallyadministered, for example, with an inert diluent or with an assimilableedible carrier, or they may be enclosed in hard or soft shell gelatincapsule, or they may be compressed into tablets, or they may beincorporated directly with the food of the diet. For oral therapeuticadministration, the active compounds may be incorporated with excipientsand used in the form of ingestible tablets, buccal tables, troches,capsules, elixirs, suspensions, syrups, wafers, and the like. Suchcompositions and preparations should contain at least 0.1% of activecompound. The percentage of the compositions and preparations may, ofcourse, be varied and may conveniently be between about 2 to about 60%of the weight of the unit. The amount of active compounds in suchtherapeutically useful compositions is such that a suitable dosage willbe obtained.

The tablets, troches, pills, capsules and the like may also contain thefollowing: a binder, as gum tragacanth, acacia, cornstarch, or gelatin;excipients, such as dicalcium phosphate; a disintegrating agent, such ascorn starch, potato starch, alginic acid and the like; a lubricant, suchas magnesium stearate; and a sweetening agent, such as sucrose, lactoseor saccharin may be added or a flavoring agent, such as peppermint, oilof wintergreen, or cherry flavoring. When the dosage unit form is acapsule, it may contain, in addition to materials of the above type, aliquid carrier. Various other materials may be present as coatings or tootherwise modify the physical form of the dosage unit. For instance,tablets, pills, or capsules may be coated with shellac, sugar or both. Asyrup of elixir may contain the active compounds sucrose as a sweeteningagent methyl and propylparabens as preservatives, a dye and flavoring,such as cherry or orange flavor. Of course, any material used inpreparing any dosage unit form should be pharmaceutically pure andsubstantially non-toxic in the amounts employed. In addition, the activecompounds may be incorporated into sustained-release preparation andformulations.

The active compounds may also be administered parenterally orintraperitoneally. Solutions of the active compounds as free base orpharmacologically acceptable salts can be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersions canalso be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial ad antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that do not produce an allergic or similar untowardreaction when administered to a human. The preparation of an aqueouscomposition that contains a protein as an active ingredient is wellunderstood in the art. Typically, such compositions are prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid prior to injectioncan also be prepared. The preparation can also be emulsified.

The composition can be formulated in a neutral or salt form.Pharmaceutically acceptable salts, include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms such as injectable solutions, drug release capsules and thelike.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, sterile aqueous media which can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage could be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biologics standards.

EXAMPLES

The following examples report the evaluation of Bb clones obtained frombiopsies and blood samples from mice infected with infectious B.burgdorferi, an in vivo selection approach for detection of antigenicvariation at the vls site and identification and characterization of thevls locus.

Example 1

Experimental Procedures

Bacterial Strains

B. burgdorferi strains B31 (ATCC 35210), Sh-2-82, and N40 wereoriginally isolated from Ixodes scapularis ticks in the state of NewYork (Burgdorfer et al., 1982; Schwan et al., 1988b; Barthold et al.,1990). These strains have been shown to be infectious in laboratoryanimals (Barthold et al., 1990; Norris et al., 1995). The high-passageB31 strain (ATCC 35210) had undergone in vitro passages for severalyears and had lost infectivity (Moody et al., 990). Nine B31 and 10Sh-2-82 passage 5 clones had been characterized according to infectivityand described previously by Norris et al. (1995). An additional ninehigh- and low-infectivity B31 clones were obtained from P. A. Rosa andT. G. Schwan of the Rocky Mountain Laboratories, Hamilton, Mont.Infectious B. afzelii ACA-1 and B. garinii IP-90 clones were obtained bysubsurface plating of organisms following isolation from experimentallyinfected C3H/HeN mice (A.G.B.). Spirochetes were cultured in BSK IImedium as described (Norris et al., 1995). The E. coli strains XL1-blueMRF′ (Stratagene, La Jolla, Calif.) and BL-21 (DE3) (Novagen, Madison,Wis.) were used for DNA cloning and fusion protein expression,respectively.

Subtractive Hybridization

Subtractive hybridization was performed according to the procedure ofSeal et al. (1992). B. burgdorferi total DNA was isolated from latelog-phase cultures (˜10¹⁰ cells) as described previously (Walker et al.,1995). Total DNA of the high-passage B. burgdorferi B31 was subjected toultrasonic disruption, and the resulting 0.5- to 1-kb fragments wereutilized as driver DNA. The driver DNA (50 μg) was then mixed with 1 μgof total DNA from the low-passage B31 digested to completion with Sau 3Al (target DNA). The target-driver DNA mixture was denatured andreannealled under the conditions described (Seal et al., 1992). Theresultant DNA mixture was ligated into BamHI-digested pBluescript II SK(−) vector (Stratagene). The ligation mixture was used to transform E.coli XL-1 blud MRF′ competent cells (Stratagene) and the transformantswere plated on Luria-Bertani (LB) agar containing 100 μg/ml ampicillin,0.5 mM isopropyl thiogalactopyranoside, and 20 μg/ml5-bromo-4-chloro-3-indolyl-D-galactoside. LB broth cultures inoculatedwith white colonies were blotted to Hybond-N⁺ nylon membranes (Amersham,Arlington Heights, Ill.) with a Bio-Dot apparatus (Bio-Rad, Hercules,Calif.) and screened by hybridization with [³²P]-labeled driver andtarget DNA. The clones that hybridized only to target probe but not todriver probe were partially sequenced using vector sequence-based T3 andT7 primers.

DNA Electrophoresis and Southern Hybridization

Total B. burgdorferi DNA was prepared in agarose inserts and separatedin 1% Fastlane agarose gels (FMC, Rockland, Me.) by pulsed-fieldelectrophoresis as described previously (Norris et al., 1995).Restriction enzyme-digested DNA fragments were separated by standardagarose gel electrophoresis (Sambrook et al., 1989). DNA bands werevisualized by ethidium bromide staining. For Southern hybridization, DNAwas blotted to Hybone-N⁺ nylon membranes by the alkaline transfer method(Sambrook et al., 1989). The blots were hybridized with [³²P]-labeledprobes at 65° C. in the presence of 1M NaCl overnight as describedpreviously (Walker et al., 1995). The blots were washed sequentially asfollows: once in 2×SSC at 65° C. for 15 min, twice in 1×RRC at 65° C.for 15 min, and twice in 0.1×SSC at room temperature for 10 min.Autoradiography was performed using X-OMAT film (Kodak, Rochester, N.Y.)with enhancing screens.

DNA Cloning and Sequence Analysis

The total plasmid DNA of B31-5A3 was prepared and treated with mung beannuclease to open the covalently linked telomeres of the linear plasmidsas described by Hinnebusch et al. (1990). The resulting plasmid DNA wasfilled in with the Klenow fragment of DNA polymerase, and an EcoRIlinker (5′-CCGGAATTCCGG-3′; SEQ ID NO:9) was ligated onto the plasmidends using T4 ligase. The preparation was then digested with EcoRI andligated into EcoRI-treated λDASH II vector (Stratagene). The recombinantphages were propagated and screened by plaque hybridization with thepJRZ53 probe according to the vector manufacturer's instructions. Lambdaphage DNA was purified by CsCl gradient purification method (Sambrook etal., 1989).

For random cloning of the λDASH-Bb12 insert, the purified bacteriophageDNA was treated with DNase I in the presence of Mn⁺⁺ and cloned into EcoRV-digested pBluescript II SK (−) as described previously (Demolis etal., 1995). The insert DNA of λDASH-Bb12 was excised from agarose gels,purified using a Qiaex II gel extraction kit (Qiagen, Chatsworth,Calif.), radiolabeled, and used as a probe to screen E. coli XL1-blueMRF′ transformants by Southern hybridization. Positive clones weresequenced as described below using T3 and T7 primers. In some instances,unsequenced regions were filled in by primer walking. The sequencedfragments were assembled using by the GELASSEMBLE program of GCG(Program Manual for the Wisconsin Package, Version 8. Genetics ComputerGroup, Madison, Wis.). High stringency settings were applied todiscriminate identical sequences from highly homologous sequences.

All plasmid and PCR™ templates were purified by Wizard columns (Promega,Madison, Wis.) and desalted through desalting columns (Amicon, Beverly,Mass.). DNA sequences were determined with an ABI377 automatic DNAsequence (Perkin-Elmer/ABI, Foster City, Calif.) in the DNA CoreLaboratory of Department of Microbiology and Molecular Genetics atUniversity of Texas Medical School at Houston. The GAP and PILEUPprograms of GCG were used to determine sequence homology (percentsimilarity and identity) and to perform multiple sequence alignments,respectively. Graphical output of alignments was prepared in partthrough the use of the BOXSHADE program (originally programmed by K.Hofmann at Bioinformatics Group, Isrec, Switzerland and M. D. Baron atthe Institute of Animal Health, Pirbright, U.K. and compiled in Pascalversion for Sun Solaris/Pascal by P. A. Stockwell at University ofOtago, Dunedin, New Zealand). Searches for sequence similarity wereperformed at the National Center for Biotechnology Information using theBLAST programs (Altschul et al., 1990).

PCR™ Techniques

All PCR™ amplifications were performed using the thermalase PCR™ kit(Amresco, Solon, Ohio) in a Minicycler from MJ Research (Watertown,Mass.). For primer pairs containing 5′-end nested sequences F4120 (SEQID NO:4) and R4121(SEQ ID NO:5), a two-step program was used as follows:96° C. for 3 min, 5 cycles of denaturation at 95° C. for 40 sec,annealing 56° C. for 40 sec, and extension at 72° C. for 2 min, followedby 30 cycles at a higher annealing at temperature at 65° C. For primerpairs without nested sequences F4064 (SEQ ID NO:6) and R4066 (SEQ IDNO:7), 35 amplification cycles of denaturation at 95° C. for 40 sec,annealing at 60° C. for 40 sec, and extension at 72° C. for 2 min wereused. The final cycles of both programs were followed by extension at72° C. for 10 min.

For RT-PCR™, total RNA was extracted from late log-phase cultures of B.burgdorferi B31-5A3 with a RNA purification kit (Amresco). The resultingRNA preparation was used to produce cDNA with the R4066 primer(5′-CTTTGCGAACGCAGACTCAGCA-3′; SEQ ID NO:10) (FIG. 2C). primer R4066,and 1 μl of the RT reaction were used for PCR™ reaction as describedabove to produce an 198-bp fragment. The PCR™ product was then clonedinto the pCR-II vector (Invitrogen, San Diego, Calif.) according to thesupplier's manual, and the resulting clones were sequenced.

GST Fusion Protein Expression

A 614-bp fragment containing the vls1 cassette was amplified by PCR™using (+) strand primer F4120 (5′-GCGGATCCAGTACGACGGGGAAACCAG-3′; SEQ IDNO:11) and (−) strand primer R4121 (5′-GCGGATCCCCTTCTCTTTCTCACCATCC-3′;SEQ ID NO:12) (FIG. 2C). For cloning purposes, the inventors' added a8-bp sequence (underlined) at the 5′-ends of both primers to createBamHI sites. The resultant PCR™ products containing the entire vls1cassette region was cloned into the BamHI site of the pGEX-2T expressionvector (Pharmacia, Piscataway, N.J.) to produce a GST fusion protein(Designated GST-Vls1) in E. coli strain BL-21(DE3) according to thesupplier's instructions. The insert sequence of the recombinant plasmidwas verified prior to use for protein expression. The fusion protein waspurified by glutathione-Sepharose 4B column (Pharmacia) according to themanufacturer's instructions.

Antibodies and Immunoblotting (Western Blotting)

Antisera against the GST-Vls1 fusion protein and GST as a control wereprepared in rabbits by immunization of rabbits with 20 μg protein incomplete Freunds adjuvant and boosting with the same amount of proteinin incomplete Freunds adjuvant at 3-week intervals (Sambrook et al.,1989). Nonspecific reactivity of the antiserum was removed by absorptionwith cell lysate of a low-infectivity B31 clone 5A2 lacking pBB28Laplasmid (FIG. 1B) as described previously (Carroll and Gherardini,1996). Antiserum against recombinant OspD was prepared in a similarmanner, and monoclonal antibody H9724 reactive with B. burgdorferiflagellum protein (Fla) was obtained as a hybridoma culture supernatantby D. D. Thomas (University of Texas Health Science Center at SanAntonio).

Late log-phase B. burgdorferi cultures were harvested by centrifugationand washed in phosphate-buffered-saline (PBS, 135 mM NaCl, 9 mM Na₂HPO₄,6 mM KH₂PO₄, pH 7.2). The organisms were resuspended at a concentrationof 10¹⁰ cells/ml in PBS. Proteins of approximately 10⁷ organisms weresubjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE), electro-transferred to PVDF membranes (Millipore, Bedford,Mass.), and detected with antibody using a ECL western blot kit fromAmersham according to the supplier's instructions.

Surface Proteolysis

Proteinase K digestion of B. burgdorferi B31-5A3 was performed asdescribed previously (Norris et al., 1992). Proteins of the treatedorganisms were separated by SDS-PAGE, electro-transferred to PVDFnitrocellulose, and reacted with antisera against GST-Vls1 or OspD orwith monoclonal antibody H9724. Reactions were visualized using the ECLwestern blot kit.

Mouse Infections

The original stock of B31-5A3 (Norris et al., 1995) was cultured in BSKII broth for 7 days, and the culture was diluted to a concentration of10⁶ cells/ml in BSK II broth. One hundred microliters of the dilutedculture (10⁵ organisms) were used to inoculate each of eight 3-week-oldfemale C3H/HeN mice by intradermal injection at the base of the tail.Each mouse was implanted with an identification microchip for follow-upsamplings during the course of infection. Four weeks after infection,the organisms were isolated by inoculating 50 μl of blood or afull-thickness biopsy (˜2 mm in diameter) of the ear into 6 ml of BSK IIbroth. All 5 ear cultures and 6 of 8 blood cultures were positive.Clonal populations of B. burgdorferi isolates from C3H/HeN mice wereobtained by subsurface plating (Norris et al., 1995). The first passagesof these cultures were frozen in BSK II medium with 20% glycerol at −70°C. as stocks for further study. The vls cassette region at theexpression site was amplified by PCR™ using primers F4120 and R4066(FIG. 2C) and sequenced using the same set of primers. Samples of thefrozen stocks (˜3 μl) were scraped from the surface, thawed, and addeddirectly into PCR™ tubes as the DNA template source to minimize possiblevariation during in vitro cultivation. Serum sample were also collectedfrom each mouse before infection and 4 weeks after initial infection andstored at −70° C. for immunoblot analysis.

Example 2

Antigenic Variation at vls Site

Bb clones isolated from ear biopsies and blood samples obtained 4 weekspost inoculation of C3H/HEN mice with the infectious B31 clone 5A3 wereevaluated. A flow diagram is provided in FIG. 8. Ear punch biopsies ˜2mm in diameter were obtained from 5 of 8 mice. The cultures were namedaccording to their source (i.e. m1e4 refers to mouse 1, ear culture, 4weeks). Clonal populations were obtained by plating on passage ofculture BSKY agar plates, and 12 colonies of each isolate were selected,cultured briefly in 2 ml BSKY medium, and frozen. Individual clones weredesignated m1e4A, m1e4B, etc. Samples of these clones were subjected toamplification of the vls cassette present in the vlsE expression site byusing primers in the “constant” regions on either side of the cassette.The resulting PCR™ products were sequenced directly.

Surprisingly, antigenic variation did not occur by substitution of theentire vls cassette at the expression site (e.g. vls1) with a single,intact “silent” vls cassette (e.g. vls2 through vls16) (see FIG. 9).Examination of 5 clones from the ear of mouse 1 (FIG. 10) indicated thatthe vlsE sequences of each clone differed from 1) the original sequence(vls1); 2) one another; and, 3) each of the silent vls cassettes. Theseresults were verified by examination of one clone from each ear or bloodisolate from the eight mice (FIG. 11). Each one of the mouse isolatesequences appeared to be comprised of a mosaic of segments from severaldifferent silent vls cassettes (between 7 and 11 in preliminaryanalyses). Sequence variability was restricted to the vls cassetteregion delimited by the ′ sequences, and in all cases the open readingframe was preserved. The vlsE cassette regions of controls consisting offive clonal populations from the inoculating culture were identical tothe original vls1 sequence, as were the sequences obtained from culturespassed 2 to 4 times in vitro (one week per passage). Therefore therearrangement process appears to be activated in vivo, and does notoccur at a rapid rate in vitro.

Example 3

Identification of the 28-kb Linear Plasmid, pBB28La

B. burgdorferi strains generally exhibit loss of infectivity following10 to 17 in vitro passages (Johnson et al., 1984; Schwan et al., 1988a;Norris et al., 1995), coinciding with the loss of plasmids (Barbour,1988; Xu et al., 1996). It was hypothesized that the decreasedinfectivity occurring during in vitro passage of Lyme disease Borreliaeis due to loss of genetic content, specifically plasmids encodingvirulence factors. Therefore, the inventors' expected to identify someof these virulence factors by directly comparing the plasmid content ofthe organisms differing in infectivity.

One of the complications involved in studying B. burgdorferi plasmids isthat many plasmids are in the 20 kb to 40 kb size range (Xu and Johnson,1995), making it difficult to resolve plasmids with similar sizes bystandard electrophoretic techniques. In addition, mutagenic techniquesand other genetic manipulation tools are in an early stage ofdevelopment in B. burgdorferi (Samuels et al., 1994; Rosa et al., 1996),thereby limiting the ability to examine the importance of these plasmidsin pathogenesis by direct genetic approaches.

To overcome these limitations, the inventors' utilized a simplesubtractive hybridization technique to enrich and eventually identifysequences present only in high-infectivity organisms.

Total DNA from a high-infectivity (low-passage) B31 strain was digestedto completion with Sau 3AI (target DNA). The target DNA was mixed with a50-fold excess of total DNA from a low-infectivity (high passage) B31derivative that had been sheared by ultrasonication (driver DNA). TheDNA mixture was denatured and allowed to reanneal. DNA fragments in theresultant DNA preparation in which the Sau 3 AI sites were regeneratedwere ligated into the BamHI site of pbluescript SK (−). A total of 63clones were isolated and screened by Southern hybridization using thetarget DNA and driver DNA as probes, respectively. Eight of these cloneshybridized with target DNA but not to driver DNA.

The inserts of the eight clones were partially sequenced using thevector-based primers, and the sequences were subjected to databasesearches for sequence similarity. One of the clones, designated pJRZ53,contained a 562-base pair (bp) Sau 3 AI fragment with a single,contiguous open reading frame. The predicted amino acid sequence of thisopen reading frame was compared to Vmps of B. hermsii, and showed 27.2%identity and 56.8% similarity to Vmp17. Based on this sequencesimilarity, the pJRZ53 insert was called a vmp-like sequence (vls). ThepJRZ53 insert exhibited a lower degree of amino acid sequence similaritywith B. burgdorferi B31 outer surface protein C (OspC) (26.6% identityand 44.5% similarity).

To identify the genomic location of the vls sequence, the pJRZ53 insertwas hybridized with Southern blots of total B31 DNA separated bypulsed-field electrophoresis. A DNA band migrating at 28 kb hybridizedto the probe (see FIG. 1B) and was determined to be a linear plasmid bytwo-dimensional agarose gel electrophoresis and restriction mapping.This vls-containing plasmid of B. burgdorferi B31 was designatedpBB28La.

Example 4

Correlation between pBB28La and Infectivity

This example illustrates the determination whether or not thevls-encoded function was required for infection. Previous studies(Norris et al., 1995), had indicated that clones of low-passage B.burgdorferi strains B31 and Sh-2-82 exhibited two distinct infectivephenotypes when tested in C3H/HeN mice. A representative high-infectiveclone had a median infectious dose (ID₅₀) of 1.8×10² organisms, whereasthe low-infective clone tested showed a much higher ID₅₀ (≧1×10⁵organisms).

It was reasoned that if the vls-encoded function is important forvirulence, all clones with the high-infective phenotype should containthe vls-containing plasmid, and loss of this plasmid would result inlow-infective phenotype. To test this hypothesis, the pJRZ53 probe washybridized with total DNA from both high- and low-infective B.burgdorferi clones. All nine B31 clones tested had a plasmid bandingpattern almost identical to each other when visualized by ethidiumbromide staining (FIG. 1A).

However, hybridization of pJRZ53 with the blot made from the same gelrevealed that all five high-infective B31 clones possessed thevls-containing pBB28La plasmid, whereas only 1 or 4 low-infective clones(B31-5A10) had this plasmid (FIG. 1B). It appears that the low-infectiveB31-5A10 clone is lacking another plasmid that correlates withinfection. Nine additional low-passage B31 clones obtained from P. A.Rosa and T. G. Schwan (Rocky Mountain Laboratories, Hamilton, Mont.)exhibited a similar pattern; all six of the high-infective clonescontained pBB28La, whereas only 1 of 3 low-infective clones containedthe plasmid, based on hybridization with the pJRZ53 probe. These resultsindicated a strong correlation of pBB28La with the high-infectivephenotype in clonal populations of B. burgdorferi B31.

To verify the correlation found in strain B31, ten previouslycharacterized clones of strain Sh-2-82 (Norris et al., 1995) wereexamined. A pBB28La homolog was detected in seven high-infective clonesbut not in three low-infective clones of strain Sh-2-82. Similar studiesrevealed the presence of a single vls-containing plasmid in infectiousB. afzelii ACA-1 and B. garinii IP-90 strains. In contrast to themultiple vmp-containing linear plasmids in B. hermsii, only onevls-containing plasmid was found in each of the Lyme disease isolatestested under the hybridization conditions employed. These vls-containingplasmids migrated consistently at ˜28 kb in agarose gels in the strainsexamined.

Out of a total of 32 clones or strains examined, all 22 clones orstrains with the high-infective phenotype contained pBB2La, and only 2of 10 low-infective clones possessed this plasmid (Table 2). TheSouthern hybridization studies indicated that the vls-containing plasmidis associated with infectivity and therefore may encode essentialvirulence factor(s).

Example 5

Cloning and Sequencing of the vls locus

A particular clonal population of B. burgdorferi B31 (clone B31-5A3) wasutilized in order to minimize clonal variation. B31-5A3 has ahigh-infective phenotype (Norris et al., 1995) and possesses the pBB28Laplasmid (FIG. 1B, lane 3). pJRZ53 was shown to hybridize with a single14-kb fragment generated by digestion of B31-5A3 plasmid DNA with EcoRI.However, treatment of the B31-5A3 plasmid DNA with PstI, Sau 3 AI, orRsaI each yielded multiple fragments ranging in size from ˜400 bp to4,000 bp that hybridized with the probe, denoting the presence ofmultiple copies of the vls sequence.

The 14-kb EcoRI fragment was cloned into λDASHII to permit a detailedanalysis of this region. The 14-kb fragment was predicted to have acovalently-closed telomere at one end. Therefore, a technique developedby Hinnebusch et al. (1992) was utilized to open the telomeric loop withmung bean nuclease and attach an EcoRI linker, thereby permittingligation into the cloning vector. A lambda clone, designated λDASH-Bb12,was isolated that contained the 14-kb B. burgdorferi DNA fragment, asconfirmed by restriction and hybridization. An internal EcoRI site wasfound to divide the λDASH-Bb12 insert into two smaller 4- and 10-kbfragments; an independently-derived clone containing the 10-kb fragmentwas also isolated during screening of the library. To verify that the4-kb and 10-kb EcoRI fragments were physically linked in the native B.burgdorferi plasmid, the region containing the internal EcoRI site wasamplified using B. burgdorferi B31-5A3 DNA as the template. Theresulting PCR™ product had a sequence identical to that of thecorresponding region of λDASH-Bb12, indicating that the 4- and 10-kbEcoRI fragments are contiguous in pBB28La. Restriction digestion of B.burgdorferi plasmid DNA at this EcoRI site was not efficient, whereascomplete cleavage was obtained consistently at the same site in theλDASH construct. Similar incomplete digestion has been observed withcertain restriction sites in B. burgdorferi chromosomal DNA (Casjens etal., 1995) and may be related to DNA modification (Hughes and Johnson,1990).

A random cloning, “shotgun” strategy was utilized to sequence nearly 10kb of the λDASH-Bb12 insert. A total of 80 random clones were sequencedusing vector-based primers. Additional sequencing reactions were carriedout to fill the gaps between the sequenced regions by primer-walking.The resulting assembled sequences had an average of 5-fold coverage. Ashort segment (˜200 bp) 1227 bp from the right telomeric end has beenrefractory to sequencing by a number of techniques. In contrast to theoverall low guanosine-cytosine (G+C) content of the B. burgdorferigenome (˜28%), the vls locus has a G+C content of 50%.

Example 6

Organization of the vls Locus

The sequence data revealed an extensive vls locus within the 10-kb EcoRIfragment consisting of an expression site (designated vlsE) and 15 vlscassettes that are highly homologous to the central portion of vlsE (SEQID NO:1 and SEQ ID NO:3). The presence of the EcoRI linker sequencebetween the insert DNA and the vector sequence defined the location ofthe right telomeric end. VlsE is located 82 bp from the right telomereof pBB281La. It possesses two unique sequences at each of the 5′ and 3′regions and a 570-bp vls cassette in the middle which was designated asthe vls1 cassette (FIG. 2B). The vls1 cassette is flanked at either endby the 17-bp direct repeat sequence 5′-GAGGGGGCTATTAAGGA-3′ (SEQ IDNO:8) encoding the amino acids EGAIK. An array of 15 vls cassettesbegins approximately 500 bp upstream of vlsE on the same plasmid (FIG.2A). The v/ls cassette and the other vls1 cassettes (vls2 through vls16)share 90.0% to 96.1% nucleotide sequence identity and 76.9% to 91.4%predicted amino acid sequence identity. The 17-bp direct repeat isconserved in nearly all of the upstream vls cassette sequences.

The vlsE gene of B. burgdorferi B31-5A3 is predicted to encode a 356amino acid protein with a molecular mass of 35,986 daltons (FIG. 2C). Aconsensus ribosome binding site and consensus −35 and −10 sigma −70-likepromoter sequences are located upstream of the predicted translationalstart site. VlsE contains a putative lipoprotein leader sequence with anapparent signal peptidase II cleavage site (FINC) (Wu and Tokunaga,1986) which resembles those of other Borrelial lipoproteins, includingOspC (Fuchs et al., 1992). Cleavage of the 18 amino acid signal peptidewould result in a mature polypeptide with a calculated molecular mass of33,956 daltons and an isoelectric point (pI) of 7.3. Except for theputative leader peptide, VlsE is predominantly hydrophilic.

VlsE shows 37.4% identity and 57.8% similarity homology at the aminoacid level and 58.8% identity at the nucleotide level to vmp17 of B.hermsii (FIG. 3A). VlsE shares a lower level of homology to B.burgdorferi ospC at both the nucleotide (41.6% identity) and amino acid(26.3% identity and 47.5% similarity) levels. The particular vlsE allelecontained in B. burgdorferi B31 clone 5A3 has been designated vlsE1, todistinguish it from variant vlsE alleles.

An additional 15 vls cassettes (474 to 594 bp in length) were identified˜500 bp upstream of vlsE (FIG. 2A and FIG. 3B, SEQ ID NO:1 and SEQ IDNO:3). These cassettes are oriented in the opposite direction to vlsEand are arranged in a head-to-tail fashion in a nearly contiguous openreading frame interrupted only by a stop codon in cassette vls11 and twoframe shifts in cassettes vls14 and vls16. None of these vls cassetteshave recognizable ribosome binding sites or promoter sequences;therefore they are thought to be nonexpressed or ‘silent’. The ends ofthe vls cassettes were defined by alignment with the vls1 cassette (FIG.3B). In general, the vls cassettes have the same 17-bp direct repeat ateither end; one exception is the joint region between vls9 and vls10,where only 10 identical nucleotides were identified. The first vdscassette (vls2) lacks the first 126 bp of the vls cassette sequence, butcontains a 55-bp sequence which is identical to the 5′ region of vlsE,coding for the last 11 amino acids of the leader peptide and the first 7amino acids of the putative mature VlsE. The vls7 cassette contains a105-bp deletion relative to vls1 in the 5′ region. The vls8 and vls10cassettes are lacking the first 54 nucleotides of the cassette. The lastcassette in this array, vls16, is truncated at the 3′ end and isfollowed by an apparent noncoding region. The 562-bp insert of pJRZ53was localized to the joining region between vls8 and vls9 by sequencecomparison.

The vls cassettes contain six highly conserved regions which areinterspersed by six variable regions (VR) at both the nucleotide andamino acid levels. FIG. 3B shows an alignment of the predicted aminoacid sequences for all 16 vls cassettes identified. Except foroccasional codon changes and the deletions mentioned previously, theconserved regions are almost identical in all cassettes. On the otherhand, the vls cassettes are distinguished from each other byconsiderable sequence variations limited predominantly to the sixvariable regions (VR-I through VR-VI). The variable regions range from21 bp (VR-VI) to 63 bp (VR-IV) in length. With exception of an insertionof a TAG stop codon in vls11 and TG insertions in vls14 and vls16resulting in frameshifts, all deletions and insertions are nucleotidetriplets, indicating preservation of the open frame. The sequencevariations at most polymorphic positions result in conservative aminoacid changes, suggesting that certain amino acids are required at thesepositions for function. Even within the six variable regions, there is aclear sequence conservation. For example, the variable sequences in VR-Iare interspersed by stretches of identical sequences ranging from 6 to 9bp, as reflected in the predicted amino acid sequences (FIG. 3B).

Example 7

Expression of VlsE

To determine whether vlsE is transcribed, reversetranscriptase-polymerase chain reaction (RT-PCR™) was utilized toamplify a 3′ region of vlsE (191 bp) from total RNA of in vitro culturedB31-5A3′. After the reverse transcriptase reaction, PCR™ amplification,and agarose electrophoresis, a band of the expected size was observed byethidium bromide staining. The RT-PCR™ product was cloned into the pCRIIvector and the recombinant plasmids were sequenced. Three independentlyderived recombinant plasmids contained DNA sequences identical to thecorresponding region of vlsE, demonstrating that vlsE is transcribed invitro. No RT-PCR™ products were observed in the agarose gel if reversetranscriptase was omitted from the reaction, confirming that the RT-PCR™products were derived from the mRNA of B31-5A3.

To identify the protein product of vlsE, an internal 614-bp fragmentcontaining vls1 was amplified by polymerase chain reaction (PCR™) andcloned into the pGEX-2T expression vector to produce aglutathione-S-transferase (GST)-vls1 fusion protein in E. coli. Rabbitantiserum against the GST-Vls1 fusion protein was used to probe proteinblots of B. burgdorferi B31-5A2 and B31-5-A3 clones. The low-infectivityclone B31-5A2 was used as a negative control for immunoblot analysis,because it lacks pBB28La (FIG. 1B, lane 2). The antiserum detected aprotein with an M_(r) of approximately 45,000 daltons in thehigh-infectivity clone B31-5A3 but not in the low-infectivity cloneB31-5A2 (FIG. 6, lanes 10 and 11). Neither the preimmune serum norantiserum against GST alone reacted with this protein. The size of theprotein identified by immunoblot analysis is larger than the predictedmolecular mass of 33,956 daltons. Attachment of a lipid moiety to theN-terminus of VlsE by signal peptidase II may contribute to the alteredelectrophoretic mobility.

Example 8

Surface Localization of VlsE

The presence of a putative lipoprotein leader peptide and the overallhydrophilic nature of VlsE raised the possibility that it is attached tothe bacterial membrane via a lipid anchor. To test this possibility, B.burgdorferi B31-5A3 was incubated in the presence of [³H]-palmitate asdescribed previously (Norris et al., 1992). VlsE was radiolabelled by[³H]-palamitate along with other B. burgdorferi lipoproteins, suggestingthat VlsE is a lipoprotein.

Exposure of viable B. burgdorferi 5A3 to proteinase K produced resultsconsistent with the surface localization of VlsE. VlsE was degraded byproteinase K in as little as 10 min (FIG. 4A), even though the organismsappeared intact by dark-field microscopy. Consistent with previous study(Norris et al., 1992), B. burgdorferi OspD protein was also removed byproteinase K treatment (FIG. 4B). In contrast, the Fla subunit of theperiplasmic flagella was not affected by proteinase K (FIG. 4C),providing further evidence that the outer membranes of the organismsremained intact during the proteinase K treatment.

Example 9

Genetic Variation at the vlsE Site

The similarity of the vls locus to the vnp system of B. hermsii promptedthe question whether genetic recombination between the expressed andsilent vls cassettes could be demonstrated in the mammalian host. Theoverall experimental design is illustrated in FIG. 5A. B. burgdorferiB31-5A3, inoculated directly from a frozen stock, was cultured for sevendays and used to intradermally inject a group of eight female C3H/HeNmice (10⁵ organisms per mouse). B. burgdorferi was re-isolated fourweeks after the initial infection. To retain the infected mice formultiple samplings at different periods of infection, only ear punchbiopsy and blood specimens were taken to culture the organisms. A totalof five ear and six blood isolates were examined. To examine possiblegenetic heterogeneity within the mouse isolates, 16 B. burgdorfericlones of each isolate were obtained by colony formation on agaroseplates and preserved by freezing. One clone from each of the isolateswas used as a source of template DNA to amplify the expressed vlscassette sequence using primers F4120 and R4066 specific for the 5′ and3′ unique regions of vlsE, respectively (FIG. 2C). The first passagefrozen stock was used to provide DNA template for PCR™ amplification toavoid possible variation during in vitro culture. The PCR™ products weresequenced directly using the same set of primers. The B. burgdorfericlones and associated sequences derived from the 4-week isolates weredesignated by a combination of mouse number (m1 to m8), tissue source (efor ear and b for blood), week post infection (4), and a clonedesignation (A to P) for the 16 clones from each isolate.

When compared with the parental vlsE of the clone B31-5A3 (allele vlsE1)inoculation, multiple base substitutions, deletions and insertions werefound within the vls cassette region of vlsE, making each allele unique.These changes resulted in numerous differences in the predicted aminoacid sequences (FIG. 5B). As found in the silent vls cassettes (FIG.3B), these mutations were primarily confined within the six variableregions. The variable sequences at almost all positions in the 11 vlsEalleles could be found in the corresponding regions of the silent vlscassettes. For example, the m1e4A and m5e4A alleles have VR-I and VR-IIidentical to vls4, whereas the VR-I and VR-III regions of m6b4A areidentical to the same regions of vls10 (FIG. 5B). These resultsindicated that changes in the original vls1 cassette have originatedfrom the silent vls cassettes via genetic recombination. In contrast,the sequences on either side of the vls cassette remained unchanged inthe 11 alleles examined (FIG. 5B).

Based on the gene conversion mechanism in vmp systems, it was initiallyhypothesized that if genetic recombination occurred at the vlsE site,the expressed vls cassette (vls1 in this case) would be replacedcompletely by a single silent cassette flanked by the 17-bp directrepeat. However, careful examination revealed that none of the 11 vlsEalleles examined were identical to any of the silent vls cassettesidentified to date. Rather, each allele appeared to be a mosaic ofsegments from several different silent vls cassettes. For instance,although m1e4A shares common sequence with vls4 throughout VR-I andVR-II, its VR-III and VR-VI are the same as vls 10 and vls2,respectively. Interestingly, the VR-IV and VR-V regions of m1e4A appearto be hybrids of portions of vls10 and vls5 and vls3 and vls5,respectively. Similar patterns can also be found in the rest of thesevlsE alleles. These observations suggested that segments, but not entireregions, of the silent vls cassettes were recombined into the expressionsite. Comparison to the silent cassette sequences at the nucleotidelevel suggested that 6 to 11 separate recombination events occurred ineach of the clones isolated from mice 4 weeks post inoculation. Thistype of combinatorial reactions could potentially result in millions ofdifferent vlsE alleles.

To determine whether the clonal populations from a single mouse alsoexhibited similar sequence variations, four additional clones of theblood and ear isolates from mouse 1 were chosen to determine the DNAsequence at the vlsE site. The five clones (m1b4A-E) of the bloodisolate had sequences identical to each other, although they showedconsiderable sequence differences from the parental vlsE as representedby m1b4A (FIG. 5B). In contrast, the sequences from the five clones ofthe ear isolate differed substantially both from the parental vls1cassette and from each other (FIG. 5C). Consistent with the 11 vlsEalleles from different isolates, the sequence variations from the sameear isolate were also concentrated in six variable regions (FIG. 5C).Each of these clones again contained a unique combination of sequencesidentical to portions of several silent vls cassettes. For example,m1e4C contained VR-I of vls12, VR-II of vls4, VR-III of vls8, and VR-IVand VR-VI of vls11. The homogeneous nature of B. burgdorferi clonesderived from the blood isolate of mouse 1 may be due to the presence ofrelatively few organisms in the blood as compared to ear biopsies,resulting essentially in cloning by limiting dilution. Alternatively,selection imposed by the host immune response in different tissueenvironments may affect diversity of vlsE variants.

The sequence variations in the clonal populations of the mouse isolatesmay also arise from background heterogeneity of the stock culture of theclone B31-5A3 occurring during in vitro culture, because the originalclone was cultured 7 days prior to the inoculation of C3H/HeN mice. Totest this possibility, the stock culture of B31-5A3 was inoculated intoBSK II medium and cultured sequentially in two in vitro passages of 7days (14 days total). PCR™ products amplified from the vlsE cassetteregion were obtained using a sample of this culture as template andeither sequenced directly or cloned into the PCR™ II vector andsequenced. Two sets of PCR™ products and four independently derivedrecombinant plasmids containing the PCR™ products all had sequencesidentical to the initial vlsE sequence. These results indicated that thesequence variations did not occur at high frequency at the vlsE siteprior to the inoculation of mice.

Example 10

Changes in Antigenicity of the VlsE Variants

The promiscuous genetic recombination at the vlsE site and the putativesurface location of VlsE suggested that sequence variations in the vlsEalleles result in changes in antigenicity.

Nine clonal populations carrying unique vlsE alleles (see FIG. 5B) weresubjected to immunoblot analysis. Although a similar amount of totalproteins were loaded into the gel as indicated by reactivity to antibodyagainst B. burgdorferi flagellin protein (FIG. 6A), these VlsE variantsexhibited a dramatic decrease in reactivity to the antiserum against theGST-Vls1 fusion protein (FIG. 6B). The mouse isolates containing m1b4Aand m3b4A alleles had bands which were weakly reactive with theantiserum (FIG. 6, lanes 2 and 5). The other clones examined exhibitedfaint bands that were visible only with a longer chemiluminescentexposures of the membrane. These reactive bands migrated at lower M_(r)sthan VlsE expressed by the parental clone B31-5A3, indicative of changesin either size or conformation. No reactive bands were observed in cloneB31-5A2, which lacks the pBB28La plasmid. The decreased reactivity ofmouse isolates with antiserum against the parental VIs1 cassette regionindicated that the sequence differences in these VlsE variants (FIG. 5B)resulted in changes in important cassette region epitopes and henceantigenic variation.

Example 11

In Vivo Expression of Vls E and Induction of Antibodies in InfectedHumans and Animals

Sera from the mice in experiments outlined in FIG. 5A were tested forreactivity with VlsE as a means of determining whether this protein isexpressed in vivo. Serum obtained from mouse 1 on 28 days postinoculation with B31-5A3 was reacted with immunoblots of 5A3 (expressingVlsE), 5A2 (lacking VlsE), the GST-Vls1 fusion protein, GST as acontrol, and two clones isolated from mouse 1 on day 28 (M1e4A andM1b4A). The results shown in FIG. 6C indicated that the C3H/HeN miceinfected with B. burgdorferi mounted a strong antibody response to VlsE.Although the prebleed serum of mouse 1 had no detectable reactivity, theserum sample collected from the same mouse 4 weeks after initialinfection with B. burgdorferi B31-5A3 reacted strongly with the GST-vls1fusion but not with GST alone, indicating expression of VlsE in themammalian host. The same serum also had a strong reactivity with theVlsE protein of B. burgdorferi B31-5A3, whereas no detectable VlsE bandwas observed with B. burgdorferi B31-5A2. In contrast, the VlsE variantM1e4A exhibited decreased reactivity when reacted with the same mouseserum as shown in FIG. 6C.

Since the C3H/HeN mice were infected with a large number (10⁵) of theorganisms (see FIG. 5A), it was possible that the antibody responseagainst VlsE had resulted from the initial inoculum. To test thispossibility, sera from white-footed mice (Peromyscus leucopus) infectedwith B. burgdorferi B31 via tick bite and from human Lyme diseasepatients were used to react with the similar immunoblots. Therepresentative results depicted in FIG. 6D showed that tick-infectedPeromyscus mice also had strong reactivity to the VlsE protein of B.burgdorferi B31-5A3 and GST-Vls1 fusion protein but not with GST alone.These results were further confirmed with sera from Lyme diseasepatients (FIG. 6E). A representative serum sample from a clinicallydiagnosed patient with early Lyme disease symptoms contained highlyreactive antibody against the VlsE protein of B31-5A3 and GST-Vls1fusion protein (FIG. 6E). Similar to the serum from the C3H/HeN mouse(FIG. 6C), the sera from the Peromyscus leucopus mouse (FIG. 6D) and theLyme disease patient (FIG. 6E) had little reactivity to the VlsE variantM1e4A. These results indicate that VlsE is expressed and is highlyimmunogenic in the mammalian host, but that genetic variation maygenerate unique VlsE variants which are no longer fully recognized bythe immune response against the parental VlsE. They also indicate thatantibodies generated against VlsE may be useful in immunodiagnosis ofLyme disease.

These results indicated that VlsE is expressed and is highly immunogenicin the mammalian host, but that genetic variation can generate uniqueVlsE variants which are no longer fully recognized by the immuneresponse against the parental VlsE. Additional experiments have shownthat some sera from Lyme disease patients also have reactivity with theGST-Vls1 fusion protein and VlsE of B. burgdorferi B31-5A3, but not withsome of the VlsE variants, thus further supporting the expression andantigenic variation of VlsE in vivo.

TABLE 5 Correlation of pBB28La with Infectivity Strains containingpBB28La/total) strains tested High-infectivity Low-infectivity Strainphenotype phenotype B. burgdoferi B31 12/12 2/7 B. burgdorferi Sh2-827/7 0/3 B. burgdorferi N40 1/1  ND^(a) B. afzelii ACA-1 1/1 ND B.garinii IP-90 1/1 ND Total 22/22 2/10 ^(a)Not determined

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48 1 1227 DNA Borrelia burgdorferi CDS (75)..(1142) 1 acctacacttgttaaaactc tctttttgag ttaagatgat aacttatact tttcattata 60 aggagacgatgaat atg aaa aaa att tca agt gca agt tta tta aca act 110 Met Lys Lys IleSer Ser Ala Ser Leu Leu Thr Thr 1 5 10 ttc ttt gtt ttt att aat tgt aaaagc caa gtt gct gat aag gac gac 158 Phe Phe Val Phe Ile Asn Cys Lys SerGln Val Ala Asp Lys Asp Asp 15 20 25 cca aca aac aaa ttt tac caa tct gtcata caa tta ggt aac gga ttt 206 Pro Thr Asn Lys Phe Tyr Gln Ser Val IleGln Leu Gly Asn Gly Phe 30 35 40 ctt gat gta ttc aca tct ttt ggt ggg ttagta gca gag gct ttt gga 254 Leu Asp Val Phe Thr Ser Phe Gly Gly Leu ValAla Glu Ala Phe Gly 45 50 55 60 ttt aaa tca gat cca aaa aaa tct gat gtaaaa acc tat ttt act act 302 Phe Lys Ser Asp Pro Lys Lys Ser Asp Val LysThr Tyr Phe Thr Thr 65 70 75 gta gct gcc aaa ttg gaa aaa aca aaa acc gatctt aat agt ttg cct 350 Val Ala Ala Lys Leu Glu Lys Thr Lys Thr Asp LeuAsn Ser Leu Pro 80 85 90 aag gaa aaa agc gat ata agt agt acg acg ggg aaacca gat agt aca 398 Lys Glu Lys Ser Asp Ile Ser Ser Thr Thr Gly Lys ProAsp Ser Thr 95 100 105 ggt tct gtt gga act gcc gtt gag ggg gct att aaggaa gtt agc gag 446 Gly Ser Val Gly Thr Ala Val Glu Gly Ala Ile Lys GluVal Ser Glu 110 115 120 ttg ttg gat aag ctg gta aaa gct gta aag aca gctgag ggg gct tca 494 Leu Leu Asp Lys Leu Val Lys Ala Val Lys Thr Ala GluGly Ala Ser 125 130 135 140 agt ggt act gct gca att gga gaa gtt gtg gctgat gct gat gct gca 542 Ser Gly Thr Ala Ala Ile Gly Glu Val Val Ala AspAla Asp Ala Ala 145 150 155 aag gtt gct gat aag gcg agt gtg aag ggg attgct aag ggg ata aag 590 Lys Val Ala Asp Lys Ala Ser Val Lys Gly Ile AlaLys Gly Ile Lys 160 165 170 gag att gtt gaa gct gct ggg ggg agt gaa aagctg aaa gct gtt gct 638 Glu Ile Val Glu Ala Ala Gly Gly Ser Glu Lys LeuLys Ala Val Ala 175 180 185 gct gct aaa ggg gag aat aat aaa ggg gca gggaag ttg ttt ggg aag 686 Ala Ala Lys Gly Glu Asn Asn Lys Gly Ala Gly LysLeu Phe Gly Lys 190 195 200 gct ggt gct gct gct cat ggg gac agt gag gctgct agc aag gcg gct 734 Ala Gly Ala Ala Ala His Gly Asp Ser Glu Ala AlaSer Lys Ala Ala 205 210 215 220 ggt gct gtt agt gct gtt agt ggg gag cagata tta agt gcg att gtt 782 Gly Ala Val Ser Ala Val Ser Gly Glu Gln IleLeu Ser Ala Ile Val 225 230 235 acg gct gct gat gcg gct gag cag gat ggaaag aag cct gag gag gct 830 Thr Ala Ala Asp Ala Ala Glu Gln Asp Gly LysLys Pro Glu Glu Ala 240 245 250 aaa aat ccg att gct gct gct att ggg gataaa gat ggg ggt gcg gag 878 Lys Asn Pro Ile Ala Ala Ala Ile Gly Asp LysAsp Gly Gly Ala Glu 255 260 265 ttt ggt cag gat gag atg aag aag gat gatcag att gct gct gct att 926 Phe Gly Gln Asp Glu Met Lys Lys Asp Asp GlnIle Ala Ala Ala Ile 270 275 280 gct ttg agg ggg atg gct aag gat gga aagttt gct gtg aag gat ggt 974 Ala Leu Arg Gly Met Ala Lys Asp Gly Lys PheAla Val Lys Asp Gly 285 290 295 300 gag aaa gag aag gct gag ggg gct attaag gga gct gct gag tct gca 1022 Glu Lys Glu Lys Ala Glu Gly Ala Ile LysGly Ala Ala Glu Ser Ala 305 310 315 gtt cgc aaa gtt tta ggg gct att actggg cta ata gga gac gcc gtg 1070 Val Arg Lys Val Leu Gly Ala Ile Thr GlyLeu Ile Gly Asp Ala Val 320 325 330 agt tcc ggg cta agg aaa gtc ggt gattca gtg aag gct gct agt aaa 1118 Ser Ser Gly Leu Arg Lys Val Gly Asp SerVal Lys Ala Ala Ser Lys 335 340 345 gaa aca cct cct gcc ttg aat aagtgatttaatt aagtgtatgg acacgactat 1172 Glu Thr Pro Pro Ala Leu Asn Lys350 355 gccctcatga ttgaggaaat agtcgagaga tatatatact aaaagataat aaata1227 2 356 PRT Borrelia burgdorferi 2 Met Lys Lys Ile Ser Ser Ala SerLeu Leu Thr Thr Phe Phe Val Phe 1 5 10 15 Ile Asn Cys Lys Ser Gln ValAla Asp Lys Asp Asp Pro Thr Asn Lys 20 25 30 Phe Tyr Gln Ser Val Ile GlnLeu Gly Asn Gly Phe Leu Asp Val Phe 35 40 45 Thr Ser Phe Gly Gly Leu ValAla Glu Ala Phe Gly Phe Lys Ser Asp 50 55 60 Pro Lys Lys Ser Asp Val LysThr Tyr Phe Thr Thr Val Ala Ala Lys 65 70 75 80 Leu Glu Lys Thr Lys ThrAsp Leu Asn Ser Leu Pro Lys Glu Lys Ser 85 90 95 Asp Ile Ser Ser Thr ThrGly Lys Pro Asp Ser Thr Gly Ser Val Gly 100 105 110 Thr Ala Val Glu GlyAla Ile Lys Glu Val Ser Glu Leu Leu Asp Lys 115 120 125 Leu Val Lys AlaVal Lys Thr Ala Glu Gly Ala Ser Ser Gly Thr Ala 130 135 140 Ala Ile GlyGlu Val Val Ala Asp Ala Asp Ala Ala Lys Val Ala Asp 145 150 155 160 LysAla Ser Val Lys Gly Ile Ala Lys Gly Ile Lys Glu Ile Val Glu 165 170 175Ala Ala Gly Gly Ser Glu Lys Leu Lys Ala Val Ala Ala Ala Lys Gly 180 185190 Glu Asn Asn Lys Gly Ala Gly Lys Leu Phe Gly Lys Ala Gly Ala Ala 195200 205 Ala His Gly Asp Ser Glu Ala Ala Ser Lys Ala Ala Gly Ala Val Ser210 215 220 Ala Val Ser Gly Glu Gln Ile Leu Ser Ala Ile Val Thr Ala AlaAsp 225 230 235 240 Ala Ala Glu Gln Asp Gly Lys Lys Pro Glu Glu Ala LysAsn Pro Ile 245 250 255 Ala Ala Ala Ile Gly Asp Lys Asp Gly Gly Ala GluPhe Gly Gln Asp 260 265 270 Glu Met Lys Lys Asp Asp Gln Ile Ala Ala AlaIle Ala Leu Arg Gly 275 280 285 Met Ala Lys Asp Gly Lys Phe Ala Val LysAsp Gly Glu Lys Glu Lys 290 295 300 Ala Glu Gly Ala Ile Lys Gly Ala AlaGlu Ser Ala Val Arg Lys Val 305 310 315 320 Leu Gly Ala Ile Thr Gly LeuIle Gly Asp Ala Val Ser Ser Gly Leu 325 330 335 Arg Lys Val Gly Asp SerVal Lys Ala Ala Ser Lys Glu Thr Pro Pro 340 345 350 Ala Leu Asn Lys 3553 7766 DNA Borrelia burgdorferi modified_base (127) R = A OR G 3gctggtgctg gtaatgctgg ggacagtgag gctgctagca aggcggctgg tgctgttagt 60gctgttagtg gggagcagat attaagtgcg attgttaagg ctgctggtga ggctgcgcag 120gatggaraga agcctgggga ggctaaaaat ccgattgctg ctgctattgg gaagggtaat 180gaggatggtg cggagtttaa ggatgagatg aagaaggatg atcagattgc tgctgctatt 240gctttgaggg ggatggctaa ggatggaaag tttgctgtga agaatgatga gaaagggaag 300gctgaggggg ctattaaggg agctggcgag ttgttggata agctggtaaa agctgtaaag 360acagctgagg gggcttcaag tggtactgct gcaattggag aagttgtggc tgatgataat 420gctgcgaagg ttgctgataa ggcgagtgtg aaggggattg ctaaggggat aaaggagatt 480gttgaagctg ctggggggag taaaaagctg aaagttgctg ctgctaaaga gggcaatgaa 540aaggcaggga agttgtttgg gaaagttgat gctgctcatg ctggggacag tgaggctgct 600agcaaggcgg ctggtgctgt tagtgctgtt agtggggagc agatattaag tgcgattgtt 660aaggctgctg gtgcggctgc tggtgatcag gagggaaaga agcctgggga tgctaaaaat 720ccgattgctg ctgctattgg gaagggtgat gcggagaatg gtgcggagtt taatcatgat 780gggatgaaga aggatgatca gattgctgct gctattgctt tgagggggat ggctaaggat 840ggaaagtttg ctgtgaagag tggtggtggt gagaaaggga aggctgaggg ggctattaag 900ggagctgctg agttgttgga taagctggta aaagctgtaa agacagctga gggggcttca 960agtggtactg atgcaattgg agaagttgtg gctaatgctg gtgctgcaaa ggttgctgat 1020aaggcgagtg tgacggggat tgctaagggg ataaaggaga ttgttgaagc tgctgggggg 1080agtgaaaagc tgaaagttgc tgctgctaca ggggagagta ataaaggggc agggaagttg 1140tttgggaagg ctggtgctgg tgctaatgct ggggacagtg aggctgctag caaggcggct 1200ggtgctgtta gtgctgttag tggggagcag atattaagtg cgattgttaa ggctgctgat 1260gcggctgatc aggagggaaa gaagcctggg gatgctacaa atccgattgc tgctgctatt 1320gggaagggta atgaggagaa tggtgcggag tttaaggatg agatgaagaa ggatgatcag 1380attgctgctg ctattgcttt gagggggatg gctaaggatg gaaagtttgc tgtgaaggat 1440ggtggtgaga aagggaaggc tgagggggct attaagggag ctgctgagtt gttggataag 1500ctggtaaaag ctgtaaagac agctgagggg gcttcaagtg gtactgatgc aattggagaa 1560gttgtggata atgctgcgaa ggctgctgat aaggcgagtg tgacggggat tgctaagggg 1620ataaaggaga ttgttgaagc tgctgggggg agtgaaaagc tgaaagttgc tgctgctaca 1680ggggagaata ataaagaggc agggaagttg tttgggaagg ctggtgctga tgctaatggg 1740gacagtgagg ctgctagcaa ggcggctggt gctgttagtg ctgttagtgg ggagcagata 1800ttaagtgcga ttgttaaggc tgcggctgct ggtgcggctg atcaggatgg agagaagcct 1860ggggatgcta aaaatccgat tgctgctgct attgggaagg gtaatgcgga tgatggtgcg 1920gattttggtg atgggatgaa gaaggatgat cagattgctg ctgctattgc tttgaggggg 1980atggctaagg atggaaagtt tgctgtgaag aaggatgaga aagggaaggc tgagggggct 2040attaagggag ctagcgagtt gttggataag ctggtaaaag ctgtaaagac agctgagggg 2100gcttcaagtg gtactgctgc aattggagaa gttgtggata atgctgcgaa ggctgctgat 2160aaggatagtg tgacggggat tgctaagggg ataaaggaga ttgttgaagc tgcagggggg 2220agtgaaaagc tgaaagttgc tgctgctaaa ggggagaata ataaaggggc agggaagttg 2280tttgggaagg ctggtgctaa tgctcatggg gacagtgagg ctgctagcaa ggcggctggt 2340gctgttagtg ctgttagtgg ggaacagata ttaagtgcga ttgttaaggc tgctggtgag 2400gctgctggtg atcaggaggg aaagaagcct gaggaggcta aaaatccgat tgctgctgct 2460attggggata aagatgggga tgcggagttt aatcaggatg ggatgaagaa ggatgatcag 2520attgctgctg ctattgcttt gagggggatg gctaaggatg gaaagtttgc tgtgaaggat 2580ggtggtgaga aagagaaggc tgagggggct attaaaggag ttagcgagtt gttggataag 2640ctggtaaaag ctgtaaagac agctgagggg gcttcaagtg gtactgctgc aattggagaa 2700gttgtggctg atgctgctaa ggttgctgat aaggcgagtg tgacggggat tgctaagggg 2760ataaaggaga ttgttgaagc tgctggggac agtgaggctg ctagcaaggc agctggtgct 2820gttagtgctg ttagtgggga gcagatatta agtgcgattg ttaaggctgc ggctgctggt 2880gcggctgagc aggatggaga gaagcctgca gaggctaaaa atccgattgc tgctgctatt 2940gggaagggtg atggggatgc ggattttggt gaggatggga tgaagaagga tgatcagatt 3000gctgctgcta ttgctttgag ggggatggct aaggatggaa agtttgctgt gaagaatgat 3060gagaaaggga aggctgaggg ggctattaag ggagctgctg caattggaga agttgtggat 3120aatgctggtg ctgcgaaggc tgctgataag gatagtgtga aggggattgc taaggggata 3180aaggagattg ttgaagctgc tggggggagt gaaaagctga aagctgctgc tgctgaaggg 3240gagaataata aaaaggcagg gaagttgttt gggaaagttg atggtgctgc tggggacagt 3300gaggctgcta gcaaggcggc tggtgctgtt agtgctgtta gtggggagca gatattaagt 3360gcgattgtta aggctgctgg tgaggctgag caggatggag agaagcctga ggatgctaaa 3420aatccgattg ctgctgctat tgggaagggt aatggggatg gtgcggagtt tgatcaggat 3480gagatgaaga aggatgatca gattgctgct gctattgctt tgagggggat ggctaaggat 3540ggaaagtttg ctgtgaaggg taataatgag aaagagaagg ctgagggggc tattaaagaa 3600gttagcgagt tgttggataa gctggtaaca gctgtaaaga cagctgaggg ggcttcaagt 3660ggtactgatg caattggaga agttgtggat aatgatgcta aggttgctga taaggcgagt 3720gtgacgggga ttgctaaggg gataaaggag attgttgaag ctgctagggg gagtgaaaag 3780ctgaaagttg ctgctgctaa agagggcaat gaaaaggcag ggaagttgtt tgggaaggct 3840ggtgctaatg ctcatgggga cagtgaggct gctagcaagg cggctggtgc tgttagtgct 3900gttagtgggg agcagatatt aagtgcgatt gttaaggctg cggatgcggc tgagcaggat 3960ggaaagaagc ctgcagatgc tacaaatccg attgctgctg ctattgggaa taaagatgag 4020gatgcggatt ttggtgatgg gatgaagaag gatgatcaga ttgctgctgc tattgctttg 4080agggggatgg ctaaggatgg aaagtttgct gtgaagggta ataatgagaa agggaaggct 4140gagggggctt caagtggtac tgatgcaatt ggagaagttg tggataatga tgcgaaggct 4200gctgataagg cgagtgtgac ggggattgct aaggggataa aggagattgt tgaagctgct 4260ggggggagtg aaaagctgaa agctgttgct gctgctacaa gggagaataa taaagaggca 4320gggaagttgt ttgggaaagt tgatgatgct catgctgggg acagtgaggc tgctagcaag 4380gcggctggtg ctgttagtgc tgttagtggg gagcagatat taagtgcgat tgttacggct 4440gcggctgctg gtgagcagga tggagagaag cctgcagagg ctacaaatcc gattgctgct 4500gctattggga agggtaatga ggatggtgcg gattttggta aggatgagat gaagaaggat 4560gatcagattg ctgctgctat tgctttgagg gggatggcta aggatggaaa gtttgctgtg 4620aagagtaatg atggtgagaa agggaaggct gagggggcta ttaaggaagt tagcgagttg 4680ttggataagc tggtaaaagc tgtaaagaca gctgaggggg cttcaagcgg tactgatgca 4740attggagaag ttgtggctaa tgctggtgct gcgaaggctg ctgataaggc gagtgtgacg 4800gggattgcta aggggataaa ggagattgtt gaagctgctg gggggagtaa aaagctgaaa 4860gctgctgctg ctgaagggga gaataataaa aaggcaggga agttgtttgg gaaggctggt 4920gctggtgctg gtgctaatgg ggacagtgag gctgctagca aggcggctgg tgctgttagt 4980gctggttagt gtggggagca gatattaagt gcgattgtta cggctgctgg tgcggctgct 5040agtgaggctg atcaggaggg aaagaagcct gcagatgcta caaatccgat tgctgctgct 5100attgggaagg gtgatgcgga gaatggtgcg gattttggtg atgggatgaa gaaggatgat 5160cagattgctg ctgctattgc tttgaggggg atggctaagg atggaaagtt tgctgtgaag 5220aatgatgatg agaaagggaa ggctgagggg gctattaagg gagctagcga gttgttggat 5280aagctggtaa cagctgtaaa gacagctgag ggggcttcaa gtggtactga tgcaattgga 5340gaagttgtgg ctgatgctgc gaaggctgct gataaggata gtgtgaaggg gattgctaag 5400gggataaagg agattgttga agctgctggg gggagtgaaa agctgaaagt tgctgctgct 5460aaagagggca atgaaaaggc agggaagttg tttgggaagg ttggtgatgc tgctcatgct 5520ggggacagtg aggctgctag caaggcggct ggtgctgtta gtgctgttag tggggagcag 5580atattaagtg cgattgttac ggctgctggt gcggctgagc aggagggaaa gaagcctgca 5640gaggctaaaa atccgattgc tgctgctatt gggaagggta atgagaatgg tgcggagttt 5700aaggatgaga tgaagaagga tgatcagatt gctgctgcta ttgctttgag ggggatggct 5760aaggatggaa agtttgctgt gaagaaggat aataatgaga aagggaaggc tgagggggct 5820attaaggaag ttagcgagtt gttggataag ctggtaacag ctgtaaagac agctgaggag 5880gcttcaagtg gtactgctgc aattggagaa gttgtggctg atgatgctgc tgcgaaggct 5940gctgataagg agagtgtgaa ggggattgct aaggggataa aggagattgt tgaagctgct 6000ggggggagta aaaagctgaa agttgctgct gctacagggg agaataataa aaaggcaggg 6060aagttgtttg ggaaagttga tgctggtaat gctggggaca gtgaggctgc tagcaaggcg 6120gctggtgctg ttagtgggga gcagatatta agtgcgattg ttaaggctgc tggtgcggct 6180gctggtgatc aggagggaaa gaagcctggg gatgctaaaa atccgattgc tgctgctatt 6240gggaagggtg atgcggagaa tggtgcggag tttgatcatg agatgaagaa ggatgatcag 6300attgctgctg ctattgcttt gagggggatg gctaaggatg gaaagtttgc tgtgaagagt 6360ggtgatgaga aagggaaggc tgagggggct attaagggag ctagcgagtt gttggataag 6420ctggtaaaag ctgtgtaaag acagctgagg gggcttcaag tggtactgat gcaattggag 6480aagttgtggc taatgatgct gctgcgaagg ttgctgataa ggagagtgtg acggggattg 6540ctaaggggat aaaggagatt gttgaagctg ctggggggag tgaaaagctg aaagttgctg 6600ctgctacaag ggagaataat aaaaaggcag ggaagttgtt tgggaaagct ggtgatgctg 6660ctaatgggga cagtgaggct gctagcaagg cggctggtgc tgttagtgct gttagtgggg 6720agcagatatt aagtgcgatt gttacggctg cagctgctgg tgcggctgag caggagggaa 6780agaagcctga ggaggctaaa aatccgattg ctgctgctat tgggaagggt aatgcggatg 6840atggtgcgga gtttaataag gaggggatga agaaggatga tcagattgct gctgctattg 6900ctttgagggg gatggctaag gatggaaagt ttgctgtgaa gagtggtggt gagaaaggga 6960aggctgaggg ggctattaaa gaagttagcg agttgttgga taagctggta acaactgtaa 7020agacagctga gggggcttca aatggtactg atgcaattgg aaaagttgtg gataataata 7080atgctgcgaa ggctgctgat aaggcgagtg tgacggggat tgctaagggg ataaaggaga 7140ttgttgaagc tgctgggggg agtagtgaaa agctgaaagc tgttgctgct gctaaagggg 7200agagcaatga aaaggcaggg aagttgtttg ggaaggctgg tgctgctgct ggggacagtg 7260aggctgctag caaggcggct ggtgctgtta gtgctgttag tggggagcag atattaagtg 7320cgattgttaa ggctgctggt gcggctgatc aggagggaaa gaagcctgag gatgctaaaa 7380atccgattgc tgctgctatt ggggataaag atgggggtgc ggagtttaat catgagatga 7440agaaggatga tcagattgct gctgctattg ctttgagggg gatggctaag gatggaaagt 7500ttgctgtgaa gagtggtggt ggtgagaaag agaaggctga gggggctatt aaagaagtta 7560gcgagttgtt ggataagctg gtaaaagctg tgtaaagaca gctgaggggg cttcaagtgg 7620tactgatgca attggagaag ttgtggctga taatagtgct gcgaaggctg ctgatgaggc 7680gagtgtgacg gggattgcta agggaataaa ggagattgtt gaagctgctg gggggagtga 7740aaagctgaaa gttgctgctg ctgcag 7766 4 16 DNA Borrelia burgdorferi 4agtacgggga aaccag 16 5 22 DNA Borrelia burgdorferi 5 ctttgcgaacgcagactcag ca 22 6 21 DNA Borrelia burgdorferi 6 agtggggaga tattaagtgc g21 7 22 DNA Borrelia burgdorferi 7 ctttgcgaac gcagactcag ca 22 8 17 DNABorrelia burgdorferi 8 gagggggcta ttaagga 17 9 12 DNA Borreliaburgdorferi 9 ccggaattcc gg 12 10 22 DNA Borrelia burgdorferi 10ctttgcgaac gcagactcag ca 22 11 27 DNA Borrelia burgdorferi 11 gcggatccagtacgacgggg aaaccag 27 12 28 DNA Borrelia burgdorferi 12 gcggatccccttctctttct caccatcc 28 13 356 PRT Borrelia burgdorferi 13 Met Lys LysIle Ser Ser Ala Ser Leu Leu Thr Thr Phe Phe Val Phe 1 5 10 15 Ile AsnCys Lys Ser Gln Val Ala Asp Lys Asp Asp Pro Thr Asn Lys 20 25 30 Phe TyrGln Ser Val Ile Gln Leu Gly Asn Gly Phe Leu Asp Val Phe 35 40 45 Thr SerPhe Gly Gly Leu Val Ala Glu Ala Phe Gly Phe Lys Ser Asp 50 55 60 Pro LysLys Ser Asp Val Lys Thr Tyr Phe Thr Thr Val Ala Ala Lys 65 70 75 80 LeuGlu Lys Thr Lys Thr Asp Leu Asn Ser Leu Pro Lys Glu Lys Ser 85 90 95 AspIle Ser Ser Thr Thr Gly Lys Pro Asp Ser Thr Gly Ser Val Gly 100 105 110Thr Ala Val Glu Gly Ala Ile Lys Glu Val Ser Glu Leu Leu Asp Lys 115 120125 Leu Val Lys Ala Val Lys Thr Ala Glu Gly Ala Ser Ser Gly Thr Ala 130135 140 Ala Ile Gly Glu Val Val Ala Asp Ala Asp Ala Ala Lys Val Ala Asp145 150 155 160 Lys Ala Ser Val Lys Gly Ile Ala Lys Gly Ile Lys Glu IleVal Glu 165 170 175 Ala Ala Gly Gly Ser Glu Lys Leu Lys Ala Val Ala AlaAla Lys Gly 180 185 190 Glu Asn Asn Lys Gly Ala Gly Lys Leu Phe Gly LysAla Gly Ala Ala 195 200 205 Ala His Gly Asp Ser Glu Ala Ala Ser Lys AlaAla Gly Ala Val Ser 210 215 220 Ala Val Ser Gly Glu Gln Ile Leu Ser AlaIle Val Thr Ala Ala Asp 225 230 235 240 Ala Ala Glu Gln Asp Gly Lys LysPro Glu Glu Ala Lys Asn Pro Ile 245 250 255 Ala Ala Ala Ile Gly Asp LysAsp Gly Gly Ala Glu Phe Gly Gln Asp 260 265 270 Glu Met Lys Lys Asp AspGln Ile Ala Ala Ala Ile Ala Leu Arg Gly 275 280 285 Met Ala Lys Asp GlyLys Phe Ala Val Lys Asp Gly Glu Lys Glu Lys 290 295 300 Ala Glu Gly AlaIle Lys Gly Ala Ala Glu Ser Ala Val Arg Lys Val 305 310 315 320 Leu GlyAla Ile Thr Gly Leu Ile Gly Asp Ala Val Ser Ser Gly Leu 325 330 335 ArgLys Val Gly Asp Ser Val Lys Ala Ala Ser Lys Glu Thr Pro Pro 340 345 350Ala Leu Asn Lys 355 14 339 PRT Borrelia burgdorferi 14 Met Arg Lys ArgIle Ser Ala Ile Ile Met Thr Leu Phe Met Val Leu 1 5 10 15 Val Ser CysAsn Ser Gly Gly Val Ala Glu Asp Pro Lys Thr Val Tyr 20 25 30 Leu Thr SerIle Ala Asn Leu Gly Lys Gly Phe Leu Asp Val Phe Val 35 40 45 Thr Phe GlyAsp Met Val Thr Gly Ala Phe Gly Ile Lys Ala Asp Thr 50 55 60 Lys Lys SerAsp Ile Gly Lys Tyr Phe Thr Asp Ile Glu Ser Thr Met 65 70 75 80 Thr SerVal Lys Lys Lys Leu Gln Asp Glu Val Ala Lys Asn Gly Asn 85 90 95 Tyr ProLys Val Lys Thr Ala Val Asp Glu Phe Val Ala Ile Leu Gly 100 105 110 LysIle Glu Lys Gly Ala Lys Glu Ala Ser Lys Gly Ala Thr Gly Asp 115 120 125Val Ile Ile Gly Asn Thr Val Lys Asn Gly Asp Ala Val Pro Gly Glu 130 135140 Ala Thr Ser Val Asn Ser Leu Val Lys Gly Ile Lys Glu Ile Val Gly 145150 155 160 Val Val Leu Lys Glu Gly Lys Ala Asp Ala Asp Ala Thr Lys AspAsp 165 170 175 Ser Lys Lys Asp Ile Gly Lys Leu Phe Thr Ala Thr Thr AspAla Asn 180 185 190 Arg Ala Asp Asn Ala Ala Ala Gln Ala Ala Ala Ala SerIle Gly Ala 195 200 205 Val Thr Gly Ala Asp Ile Leu Gln Ala Ile Val GlnSer Lys Glu Asn 210 215 220 Pro Val Ala Asn Ser Thr Asp Gly Ile Glu LysAla Thr Asp Ala Ala 225 230 235 240 Glu Ile Ala Val Ala Pro Ala Lys AspAsn Lys Lys Glu Ile Lys Asp 245 250 255 Gly Ala Lys Lys Asp Ala Val IleAla Ala Gly Ile Ala Leu Arg Ala 260 265 270 Met Ala Lys Asn Gly Thr PheSer Ile Lys Asn Asn Glu Asp Ala Ala 275 280 285 Val Thr Thr Ile Asn SerAla Ala Ala Ser Ala Val Asn Lys Ile Leu 290 295 300 Ser Thr Leu Ile IleAla Ile Arg Asn Thr Val Asp Ser Gly Leu Lys 305 310 315 320 Thr Ile AsnGlu Ala Leu Ala Thr Val Lys Gln Glu Asp Lys Ser Val 325 330 335 Glu AlaThr 15 190 PRT Borrelia burgdorferi 15 Glu Gly Ala Ile Lys Glu Val SerGlu Leu Leu Asp Lys Leu Val Lys 1 5 10 15 Ala Val Lys Thr Ala Glu GlyAla Ser Ser Gly Thr Ala Ala Ile Gly 20 25 30 Glu Val Val Ala Asp Ala AspAla Ala Lys Val Ala Asp Lys Ala Ser 35 40 45 Val Lys Gly Ile Ala Lys GlyIle Lys Glu Ile Val Glu Ala Ala Gly 50 55 60 Gly Ser Glu Lys Leu Lys AlaVal Ala Ala Ala Lys Gly Glu Asn Asn 65 70 75 80 Lys Gly Ala Gly Lys LeuPhe Gly Lys Ala Gly Ala Ala Ala His Gly 85 90 95 Asp Ser Glu Ala Ala SerLys Ala Ala Gly Ala Val Ser Ala Val Ser 100 105 110 Gly Glu Gln Ile LeuSer Ala Ile Val Thr Ala Ala Asp Ala Ala Glu 115 120 125 Gln Asp Gly LysLys Pro Glu Glu Ala Lys Asn Pro Ile Ala Ala Ala 130 135 140 Ile Gly AspLys Asp Gly Gly Ala Glu Phe Gly Gln Asp Glu Met Lys 145 150 155 160 LysAsp Asp Gln Ile Ala Ala Ala Ile Ala Leu Arg Gly Met Ala Lys 165 170 175Asp Gly Lys Phe Ala Val Lys Asp Gly Glu Lys Glu Lys Ala 180 185 190 16169 PRT Borrelia burgdorferi 16 Met Thr Asn Pro Ser Thr Ala Thr Leu LeuThr Thr Phe Phe Val Phe 1 5 10 15 Ile Asn Cys Lys Ser Gln Val Ala AspLys Ala Ser Val Thr Gly Ile 20 25 30 Ala Lys Gly Ile Lys Glu Ile Val GluAla Ala Gly Gly Ser Glu Lys 35 40 45 Leu Lys Val Ala Ala Ala Glu Gly GluAsn Asn Glu Lys Ala Gly Lys 50 55 60 Leu Phe Gly Lys Ala Gly Ala Gly AsnAla Gly Asp Ser Glu Ala Ala 65 70 75 80 Ser Lys Ala Ala Gly Ala Val SerAla Val Ser Gly Glu Gln Ile Leu 85 90 95 Ser Ala Ile Val Lys Ala Ala GlyGlu Ala Ala Gln Asp Gly Lys Lys 100 105 110 Pro Gly Glu Ala Lys Asn ProIle Ala Ala Ala Ile Gly Lys Gly Asn 115 120 125 Glu Asp Gly Ala Glu PheLys Asp Glu Met Lys Lys Asp Asp Gln Ile 130 135 140 Ala Ala Ala Ile AlaLeu Arg Gly Met Ala Lys Asp Gly Lys Phe Ala 145 150 155 160 Val Lys AsnAsp Glu Lys Gly Lys Ala 165 17 195 PRT Borrelia burgdorferi 17 Glu GlyAla Ile Lys Gly Ala Gly Glu Leu Leu Asp Lys Leu Val Lys 1 5 10 15 AlaVal Lys Thr Ala Glu Gly Ala Ser Ser Gly Thr Ala Ala Ile Gly 20 25 30 GluVal Val Ala Asp Asp Asn Ala Ala Lys Val Ala Asp Lys Ala Ser 35 40 45 ValLys Gly Ile Ala Lys Gly Ile Lys Glu Ile Val Glu Ala Ala Gly 50 55 60 GlySer Lys Lys Leu Lys Val Ala Ala Ala Lys Gly Glu Gly Asn Glu 65 70 75 80Lys Ala Gly Lys Leu Phe Gly Lys Val Asp Ala Ala His Ala Gly Asp 85 90 95Ser Glu Ala Ala Ser Lys Ala Ala Gly Ala Val Ser Ala Val Ser Gly 100 105110 Glu Gln Ile Leu Ser Ala Ile Val Lys Ala Ala Gly Ala Ala Ala Gly 115120 125 Asp Gln Glu Gly Lys Lys Pro Gly Asp Ala Lys Asn Pro Ile Ala Ala130 135 140 Ala Ile Gly Lys Gly Asp Ala Glu Asn Gly Ala Glu Phe Asn HisAsp 145 150 155 160 Gly Met Lys Lys Asp Asp Gln Ile Ala Ala Ala Ile AlaLeu Arg Gly 165 170 175 Met Ala Lys Asp Gly Lys Phe Ala Val Lys Ser GlyGly Gly Glu Lys 180 185 190 Gly Lys Ala 195 18 179 PRT Borreliaburgdorferi 18 Glu Gly Ala Ile Lys Gly Ala Ala Glu Leu Leu Asp Lys LeuVal Lys 1 5 10 15 Ala Val Lys Thr Ala Glu Gly Ala Ser Ser Gly Thr AspAla Ile Gly 20 25 30 Glu Val Val Ala Asn Ala Gly Ala Ala Lys Val Ala AspLys Ala Ser 35 40 45 Val Thr Gly Ile Ala Lys Gly Ile Lys Glu Ile Val GluAla Ala Gly 50 55 60 Gly Ser Glu Lys Leu Lys Val Ala Ala Ala Thr Gly GluSer Gly Ala 65 70 75 80 Asn Ala Gly Asp Ser Glu Ala Ala Ser Lys Ala AlaGly Ala Val Ser 85 90 95 Ala Val Ser Gly Glu Gln Ile Leu Ser Ala Ile ValLys Ala Ala Asp 100 105 110 Ala Ala Asp Gln Glu Gly Lys Lys Pro Gly AspAla Thr Asn Pro Ile 115 120 125 Ala Ala Ala Ile Gly Lys Gly Asn Glu GluAsn Gly Ala Glu Phe Lys 130 135 140 Asp Glu Met Lys Lys Asp Asp Gln IleAla Ala Ala Ile Ala Leu Arg 145 150 155 160 Gly Met Ala Lys Asp Gly LysPhe Ala Val Lys Asp Gly Gly Glu Lys 165 170 175 Gly Lys Ala 19 190 PRTBorrelia burgdorferi 19 Glu Gly Ala Ile Lys Gly Ala Ala Glu Leu Leu AspLys Leu Val Lys 1 5 10 15 Ala Val Lys Thr Ala Glu Gly Ala Ser Ser GlyThr Asp Ala Ile Gly 20 25 30 Glu Val Val Asp Asn Ala Ala Lys Ala Ala AspLys Ala Ser Val Thr 35 40 45 Gly Ile Ala Lys Gly Ile Lys Glu Ile Val GluAla Ala Gly Gly Ser 50 55 60 Glu Lys Leu Lys Val Ala Ala Ala Thr Gly GluAsn Asn Lys Glu Ala 65 70 75 80 Gly Lys Leu Phe Gly Lys Ala Gly Ala AspAla Asn Gly Asp Ser Glu 85 90 95 Ala Ala Ser Lys Ala Ala Gly Ala Val SerAla Val Ser Gly Glu Gln 100 105 110 Ile Leu Ser Ala Ile Val Lys Ala AlaAla Ala Gly Ala Ala Asp Gln 115 120 125 Asp Gly Glu Lys Pro Gly Asp AlaLys Asn Pro Ile Ala Ala Ala Ile 130 135 140 Gly Lys Gly Asn Ala Asp AspGly Ala Asp Phe Gly Asp Gly Met Lys 145 150 155 160 Lys Asp Asp Gln IleAla Ala Ala Ile Ala Leu Arg Gly Met Ala Lys 165 170 175 Asp Gly Lys PheAla Val Lys Lys Asp Glu Lys Gly Lys Ala 180 185 190 20 189 PRT Borreliaburgdorferi 20 Glu Gly Ala Ile Lys Gly Ala Ser Glu Leu Leu Asp Lys LeuVal Lys 1 5 10 15 Ala Val Lys Thr Ala Glu Gly Ala Ser Ser Gly Thr AlaAla Ile Gly 20 25 30 Glu Val Val Asp Asn Ala Ala Lys Ala Ala Asp Lys AspSer Val Thr 35 40 45 Gly Ile Ala Lys Gly Ile Lys Glu Ile Val Glu Ala AlaGly Gly Ser 50 55 60 Glu Lys Leu Lys Val Ala Ala Ala Lys Gly Glu Asn AsnLys Gly Ala 65 70 75 80 Gly Lys Leu Phe Gly Lys Ala Gly Ala Asn Ala HisGly Asp Ser Glu 85 90 95 Ala Ala Ser Lys Ala Ala Gly Ala Val Ser Ala ValSer Gly Glu Gln 100 105 110 Ile Leu Ser Ala Ile Val Lys Ala Ala Gly GluAla Ala Gly Asp Gln 115 120 125 Glu Gly Lys Lys Pro Glu Glu Ala Lys AsnPro Ile Ala Ala Ala Ile 130 135 140 Gly Lys Asp Gly Asp Ala Glu Asn GlyGln Gly Glu Met Lys Lys Asp 145 150 155 160 Asp Gln Ile Ala Ala Ala IleAla Leu Arg Gly Met Ala Lys Asp Gly 165 170 175 Lys Phe Ala Val Lys AspGly Gly Glu Lys Glu Lys Ala 180 185 21 158 PRT Borrelia burgdorferi 21Glu Gly Ala Ile Lys Gly Val Ser Glu Leu Leu Asp Lys Leu Val Lys 1 5 1015 Ala Val Lys Thr Ala Glu Gly Ala Ser Ser Gly Thr Ala Ala Ile Gly 20 2530 Glu Val Val Ala Asp Ala Ala Lys Val Ala Asp Lys Ala Ser Val Thr 35 4045 Gly Ile Ala Lys Gly Ile Lys Glu Ile Val Glu Ala Ala Gly Asp Ser 50 5560 Glu Ala Ala Ser Lys Ala Ala Gly Ala Val Ser Ala Val Ser Gly Glu 65 7075 80 Gln Ile Leu Ser Ala Ile Val Lys Ala Ala Ala Ala Gly Ala Ala Glu 8590 95 Gln Asp Gly Glu Lys Pro Ala Glu Ala Lys Asn Pro Ile Ala Ala Ala100 105 110 Ile Gly Lys Gly Asp Gly Asp Ala Asp Phe Gly Glu Asp Gly MetLys 115 120 125 Lys Asp Asp Gln Ile Ala Ala Ala Ile Ala Leu Arg Gly MetAla Lys 130 135 140 Asp Gly Lys Phe Ala Val Lys Asn Asp Glu Lys Gly LysAla 145 150 155 22 169 PRT Borrelia burgdorferi 22 Glu Gly Ala Ile LysGly Ala Ala Ala Ile Gly Glu Val Val Asp Asn 1 5 10 15 Ala Gly Ala AlaLys Ala Ala Asp Lys Asp Ser Val Lys Gly Ile Ala 20 25 30 Lys Gly Ile LysGlu Ile Val Glu Ala Ala Gly Gly Ser Glu Lys Leu 35 40 45 Lys Ala Ala AlaAla Glu Gly Glu Asn Asn Lys Lys Ala Gly Lys Leu 50 55 60 Phe Gly Lys ValAsp Gly Ala Ala Gly Asp Ser Glu Ala Ala Ser Lys 65 70 75 80 Ala Ala GlyAla Val Ser Ala Val Ser Gly Glu Gln Ile Leu Ser Ala 85 90 95 Ile Val LysAla Ala Gly Glu Ala Glu Gln Asp Glu Lys Lys Pro Glu 100 105 110 Asp AlaLys Asn Pro Ile Ala Ala Ala Ile Gly Lys Gly Asn Gly Asp 115 120 125 GlyAla Glu Phe Asp Gln Asp Glu Met Lys Lys Asp Asp Gln Ile Ala 130 135 140Ala Ala Ile Ala Leu Arg Gly Met Ala Lys Asp Gly Lys Phe Ala Val 145 150155 160 Lys Gly Asn Asn Glu Lys Glu Lys Ala 165 23 187 PRT Borreliaburgdorferi 23 Glu Gly Ala Ile Lys Glu Val Ser Glu Leu Leu Asp Lys LeuVal Thr 1 5 10 15 Ala Val Lys Thr Ala Glu Gly Ala Ser Ser Gly Thr AspAla Ile Gly 20 25 30 Glu Val Val Asp Asn Asp Ala Lys Val Ala Asp Lys AlaSer Val Thr 35 40 45 Gly Ile Ala Lys Gly Ile Lys Glu Ile Val Glu Ala AlaArg Gly Ser 50 55 60 Glu Lys Leu Lys Ala Val Ala Ala Ala Lys Glu Gly AsnGlu Lys Ala 65 70 75 80 Gly Lys Leu Phe Gly Lys Ala Gly Ala Asn Ala HisGly Asp Ser Glu 85 90 95 Ala Ala Ser Lys Ala Ala Gly Ala Val Ser Ala ValSer Gly Glu Gln 100 105 110 Ile Leu Ser Ala Ile Val Lys Ala Ala Asp AlaAla Glu Gln Asp Gly 115 120 125 Lys Lys Pro Ala Asp Ala Thr Asn Pro IleAla Ala Ala Ile Gly Asn 130 135 140 Lys Asp Glu Asp Ala Asp Phe Gly AspGly Met Lys Lys Asp Asp Gln 145 150 155 160 Ile Ala Ala Ala Ile Ala LeuArg Gly Met Ala Lys Asp Gly Lys Phe 165 170 175 Ala Val Lys Gly Asn AsnGlu Lys Gly Lys Ala 180 185 24 169 PRT Borrelia burgdorferi 24 Glu GlyAla Ser Ser Gly Thr Asp Ala Ile Gly Glu Val Val Asp Asn 1 5 10 15 AspAla Lys Ala Ala Asp Lys Ala Ser Val Thr Gly Ile Ala Lys Gly 20 25 30 IleLys Glu Ile Val Glu Ala Ala Gly Gly Ser Glu Lys Leu Lys Ala 35 40 45 ValAla Ala Ala Thr Arg Glu Asn Asn Lys Glu Ala Gly Lys Leu Phe 50 55 60 GlyLys Val Asp Asp Ala His Ala Gly Asp Ser Glu Ala Ala Ser Lys 65 70 75 80Ala Ala Gly Ala Val Ser Ala Val Ser Gly Glu Gln Ile Leu Ser Ala 85 90 95Ile Val Thr Ala Ala Ala Ala Gly Glu Gln Asp Gly Glu Lys Pro Ala 100 105110 Glu Ala Thr Asn Pro Ile Ala Ala Ala Ile Gly Lys Gly Asn Glu Asp 115120 125 Gly Ala Asp Phe Gly Lys Asp Glu Met Lys Lys Asp Asp Gln Ile Ala130 135 140 Ala Ala Ile Ala Leu Arg Gly Met Ala Lys Asp Gly Lys Phe AlaVal 145 150 155 160 Lys Ser Asn Asp Gly Lys Gly Lys Ala 165 25 197 PRTBorrelia burgdorferi 25 Glu Gly Ala Ile Lys Glu Val Ser Glu Leu Leu AspLys Leu Val Lys 1 5 10 15 Ala Val Lys Thr Ala Glu Gly Ala Ser Ser GlyThr Asp Ala Ile Gly 20 25 30 Glu Val Val Ala Asn Ala Gly Ala Ala Lys AlaAla Asp Lys Ala Ser 35 40 45 Val Thr Gly Ile Ala Lys Gly Ile Lys Glu IleVal Glu Ala Ala Gly 50 55 60 Gly Ser Lys Lys Leu Lys Ala Ala Ala Ala GluGly Glu Asn Asn Lys 65 70 75 80 Lys Ala Gly Lys Leu Phe Gly Lys Ala GlyAla Gly Ala Gly Ala Asn 85 90 95 Gly Asp Ser Glu Ala Ala Ser Lys Ala AlaGly Ala Val Ser Ala Gly 100 105 110 Cys Gly Glu Gln Ile Leu Ser Ala IleVal Thr Ala Ala Gly Ala Ala 115 120 125 Ala Ser Glu Ala Asp Gln Glu GlyLys Lys Pro Ala Asp Ala Thr Asn 130 135 140 Pro Ile Ala Ala Ala Ile GlyLys Gly Asp Ala Glu Asn Gly Ala Asp 145 150 155 160 Phe Gly Asp Gly MetLys Lys Asp Asp Gln Ile Ala Ala Ala Ile Ala 165 170 175 Leu Arg Gly MetAla Lys Asp Gly Lys Phe Ala Val Lys Asn Asp Asp 180 185 190 Glu Lys GlyLys Ala 195 26 189 PRT Borrelia burgdorferi 26 Glu Gly Ala Ile Lys GlyAla Ser Glu Leu Leu Asp Lys Leu Val Thr 1 5 10 15 Ala Val Lys Thr AlaGlu Gly Ala Ser Ser Gly Thr Asp Ala Ile Gly 20 25 30 Glu Val Val Ala AspAla Ala Lys Ala Ala Asp Lys Asp Ser Val Lys 35 40 45 Gly Ile Ala Lys GlyIle Lys Glu Ile Val Glu Ala Ala Gly Gly Ser 50 55 60 Glu Lys Leu Lys ValAla Ala Ala Lys Glu Gly Asn Glu Lys Ala Gly 65 70 75 80 Lys Leu Phe GlyLys Val Gly Asp Ala Ala His Ala Gly Asp Ser Glu 85 90 95 Ala Ala Ser LysAla Ala Gly Ala Val Ser Ala Val Ser Gly Glu Gln 100 105 110 Ile Leu SerAla Ile Val Thr Ala Ala Gly Ala Ala Glu Gln Glu Gly 115 120 125 Lys LysPro Ala Glu Ala Lys Asn Pro Ile Ala Ala Ala Ile Gly Lys 130 135 140 GlyAsn Glu Asn Gly Ala Glu Phe Lys Asp Glu Met Lys Lys Asp Asp 145 150 155160 Gln Ile Ala Ala Ala Ile Ala Leu Arg Gly Met Ala Lys Asp Gly Lys 165170 175 Phe Ala Val Lys Lys Asp Asn Asn Glu Lys Gly Lys Ala 180 185 27190 PRT Borrelia burgdorferi 27 Glu Gly Ala Ile Lys Glu Val Ser Glu LeuLeu Asp Lys Leu Val Thr 1 5 10 15 Ala Val Lys Thr Ala Glu Glu Ala SerSer Gly Thr Ala Ala Ile Gly 20 25 30 Glu Val Val Ala Asp Asp Ala Ala AlaLys Ala Ala Asp Lys Glu Ser 35 40 45 Val Lys Gly Ile Ala Lys Gly Ile LysGlu Ile Val Glu Ala Ala Gly 50 55 60 Gly Ser Lys Lys Leu Lys Val Ala AlaAla Thr Gly Glu Asn Asn Lys 65 70 75 80 Lys Ala Gly Lys Leu Phe Gly LysVal Asp Ala Gly Asn Ala Gly Asp 85 90 95 Ser Glu Ala Ala Ser Lys Ala AlaGly Ala Val Ser Gly Glu Gln Ile 100 105 110 Leu Ser Ala Ile Val Lys AlaAla Gly Ala Ala Ala Gly Asp Asp Glu 115 120 125 Lys Lys Pro Gly Asp AlaLys Asn Pro Ile Ala Ala Ala Ile Gly Lys 130 135 140 Gly Asp Ala Glu AsnGly Ala Glu Phe Asp His Glu Met Lys Lys Asp 145 150 155 160 Asp Gln IleAla Ala Ala Ile Ala Leu Arg Gly Met Ala Lys Asp Gly 165 170 175 Lys PheAla Val Lys Ser Asp Gly Asp Glu Lys Gly Lys Ala 180 185 190 28 194 PRTBorrelia burgdorferi 28 Glu Gly Ala Ile Lys Gly Ala Ser Glu Leu Leu AspLys Leu Val Lys 1 5 10 15 Ala Val Lys Thr Ala Glu Gly Ala Ser Ser GlyThr Asp Ala Ile Gly 20 25 30 Glu Val Val Ala Asn Asp Ala Ala Ala Lys ValAla Asp Lys Glu Ser 35 40 45 Val Thr Gly Ile Ala Lys Gly Ile Lys Glu IleVal Glu Ala Ala Gly 50 55 60 Gly Ser Glu Lys Leu Lys Val Ala Ala Ala ThrArg Glu Asn Asn Lys 65 70 75 80 Lys Ala Gly Lys Leu Phe Gly Lys Ala GlyAsp Ala Ala Asn Gly Asp 85 90 95 Ser Glu Ala Ala Ser Lys Ala Ala Gly AlaVal Ser Ala Val Ser Gly 100 105 110 Glu Gln Ile Leu Ser Ala Ile Val ThrAla Ala Ala Ala Gly Ala Ala 115 120 125 Glu Gln Glu Gly Lys Lys Pro GluGlu Ala Lys Asn Pro Ile Ala Ala 130 135 140 Ala Ile Gly Lys Gly Asn AlaAsp Asp Gly Ala Glu Phe Asn Lys Glu 145 150 155 160 Gly Met Lys Lys AspAsp Gln Ile Ala Ala Ala Ile Ala Leu Arg Gly 165 170 175 Met Ala Lys AspGly Lys Phe Ala Val Lys Ser Gly Gly Glu Lys Gly 180 185 190 Lys Ala 29190 PRT Borrelia burgdorferi 29 Glu Gly Ala Ile Lys Glu Val Ser Glu LeuLeu Asp Lys Leu Val Thr 1 5 10 15 Thr Val Lys Thr Ala Glu Gly Ala SerAsn Gly Thr Asp Ala Ile Gly 20 25 30 Lys Val Val Asp Asn Asn Asn Ala AlaLys Ala Ala Asp Lys Ala Ser 35 40 45 Val Thr Gly Ile Ala Lys Gly Ile LysGlu Ile Val Glu Ala Ala Gly 50 55 60 Gly Ser Ser Glu Lys Leu Lys Ala ValAla Ala Ala Lys Gly Glu Ser 65 70 75 80 Asn Glu Lys Ala Gly Lys Leu PheGly Lys Ala Gly Ala Ala Ala Gly 85 90 95 Asp Ser Glu Ala Ala Ser Lys AlaAla Gly Ala Val Ser Ala Val Ser 100 105 110 Gly Glu Gln Ile Leu Ser AlaIle Val Lys Ala Ala Gly Ala Ala Asp 115 120 125 Gln Glu Gly Lys Lys ProGlu Glu Asp Lys Asn Pro Ile Ala Ala Ala 130 135 140 Ile Gly Asp Lys AspGly Gly Ala Glu Phe Asn His Glu Met Lys Lys 145 150 155 160 Asp Asp GlnIle Ala Ala Ala Ile Ala Leu Arg Gly Met Ala Lys Asp 165 170 175 Gly LysPhe Ala Val Lys Ser Gly Gly Glu Lys Glu Lys Ala 180 185 190 30 157 PRTBorrelia burgdorferi 30 Glu Gly Ala Ile Lys Glu Val Ser Glu Leu Leu AspLys Leu Val Lys 1 5 10 15 Ala Val Lys Thr Ala Glu Gly Ala Ser Ser GlyThr Asp Ala Ile Gly 20 25 30 Glu Val Val Ala Asp Asn Ser Ala Ala Lys AlaAla Asp Glu Ala Ser 35 40 45 Val Thr Gly Ile Ala Lys Gly Ile Lys Glu IleVal Glu Ala Ala Gly 50 55 60 Gly Ser Glu Lys Leu Lys Val Ala Ala Ala AlaGly Glu Asn Asn Lys 65 70 75 80 Lys Ala Gly Lys Leu Phe Gly Lys Val AspAsn Ala Asn Ala Gly Asp 85 90 95 Ser Glu Ala Ala Ser Lys Ala Ala Gly AlaVal Ser Gly Glu Gln Ile 100 105 110 Leu Ser Ala Ile Val Lys Ala Ala GlyGlu Ala Glu Gln Asp Gly Glu 115 120 125 Lys Pro Gly Glu Ala Thr Lys GlyAsp Glu Asp Ala Asp Phe Gly Asn 130 135 140 Glu Met Lys Lys Asp Gly LysPhe Ala Ile Leu Thr Phe 145 150 155 31 212 PRT Borrelia burgdorferi 31Ser Thr Thr Gly Lys Pro Asp Ser Thr Gly Ser Val Gly Thr Ala Val 1 5 1015 Glu Gly Ala Ile Lys Glu Val Ser Glu Leu Leu Asp Lys Leu Val Lys 20 2530 Ala Val Lys Thr Ala Glu Gly Ala Ser Ser Gly Thr Ala Ala Ile Gly 35 4045 Glu Val Val Ala Asp Ala Asp Ala Ala Lys Val Ala Asp Lys Ala Ser 50 5560 Val Lys Gly Ile Ala Lys Gly Ile Lys Glu Ile Val Glu Ala Ala Gly 65 7075 80 Gly Ser Glu Lys Leu Lys Ala Val Ala Ala Ala Lys Gly Glu Asn Asn 8590 95 Lys Gly Ala Gly Lys Leu Phe Gly Lys Ala Gly Ala Ala Ala His Gly100 105 110 Asp Ser Glu Ala Ala Ser Lys Ala Ala Gly Ala Val Ser Ala ValSer 115 120 125 Gly Glu Gln Ile Leu Ser Ala Ile Val Thr Ala Ala Asp AlaAla Glu 130 135 140 Gln Asp Gly Lys Lys Pro Glu Glu Ala Lys Asn Pro IleAla Ala Ala 145 150 155 160 Ile Gly Asp Lys Asp Gly Gly Ala Glu Phe GlyGln Asp Glu Met Lys 165 170 175 Lys Asp Asp Gln Ile Ala Ala Ala Ile AlaLeu Arg Gly Met Ala Lys 180 185 190 Asp Gly Lys Phe Ala Val Lys Asp GlyGlu Lys Glu Lys Ala Glu Gly 195 200 205 Ala Ile Lys Gly 210 32 212 PRTBorrelia burgdorferi 32 Ser Thr Thr Gly Lys Pro Asp Ser Thr Gly Ser ValGly Thr Ala Val 1 5 10 15 Glu Gly Ala Ile Lys Glu Val Ser Glu Leu LeuAsp Lys Leu Val Lys 20 25 30 Ala Val Lys Thr Ala Glu Gly Ala Ser Ser GlyThr Asp Ala Ile Gly 35 40 45 Glu Val Val Ala Asn Ala Gly Ala Ala Lys ValAla Asp Lys Ala Ser 50 55 60 Val Thr Gly Ile Ala Lys Gly Ile Lys Glu IleVal Glu Ala Ala Gly 65 70 75 80 Gly Ser Glu Lys Leu Lys Val Ala Ala AlaThr Gly Glu Ser Asn Lys 85 90 95 Gly Ala Gly Lys Leu Phe Gly Lys Val AspAsp Ala His Ala Gly Asp 100 105 110 Ser Glu Ala Ala Ser Lys Ala Ala GlyAla Val Ser Ala Val Ser Gly 115 120 125 Glu Gln Ile Leu Ser Ala Ile ValThr Ala Ala Ala Ala Gly Glu Gln 130 135 140 Asp Gly Glu Lys Pro Glu GlyAsp Lys Asn Pro Ile Ala Ala Ala Ile 145 150 155 160 Gly Lys Gly Asp AlaAsp Asp Gly Ala Asp Phe Gly Asp Gly Met Lys 165 170 175 Lys Asp Asp GlnIle Ala Ala Ala Ile Ala Leu Arg Gly Met Ala Lys 180 185 190 Asp Gly LysPhe Ala Val Lys Asn Asp Glu Lys Gly Lys Ala Glu Gly 195 200 205 Ala IleLys Gly 210 33 209 PRT Borrelia burgdorferi 33 Ser Thr Thr Gly Lys ProAsp Ser Thr Gly Ser Val Gly Thr Ala Val 1 5 10 15 Glu Gly Ala Ile LysGlu Val Ser Glu Leu Leu Asp Lys Leu Val Lys 20 25 30 Ala Val Lys Thr AlaGlu Gly Ala Ser Ser Gly Thr Asp Ala Ile Gly 35 40 45 Glu Val Val Asp AsnAla Ala Lys Ala Ala Asp Lys Ala Ser Val Thr 50 55 60 Gly Ile Ala Lys GlyIle Lys Glu Ile Val Glu Ala Ala Gly Gly Ser 65 70 75 80 Glu Lys Leu LysVal Ala Ala Ala Thr Gly Glu Ser Asn Lys Gly Ala 85 90 95 Gly Lys Leu PheGly Lys Ala Gly Ala Gly Ala Asn Gly Asp Ser Glu 100 105 110 Ala Ala SerLys Ala Ala Gly Ala Val Ser Ala Val Ser Gly Glu Gln 115 120 125 Ile LeuSer Ala Ile Val Lys Ala Ala Gly Ala Ala Asp Gln Asp Gly 130 135 140 GluLys Pro Gly Asp Ala Lys Asn Pro Ile Ala Ala Ala Ile Gly Lys 145 150 155160 Gly Asp Gly Asp Ala Glu Phe Asp Gln Asp Glu Met Lys Lys Asp Asp 165170 175 Gln Ile Ala Ala Ala Ile Ala Leu Arg Gly Met Ala Lys Asp Gly Lys180 185 190 Phe Ala Val Lys Asn Asp Glu Lys Gly Lys Ala Glu Gly Ala IleLys 195 200 205 Gly 34 214 PRT Borrelia burgdorferi 34 Ser Thr Thr GlyLys Pro Asp Ser Thr Gly Ser Val Gly Thr Ala Val 1 5 10 15 Glu Gly AlaIle Lys Glu Val Ser Glu Leu Leu Asp Lys Leu Val Lys 20 25 30 Ala Val LysThr Ala Glu Gly Ala Ser Ser Gly Thr Ala Ala Ile Gly 35 40 45 Glu Val ValAsp Asn Asp Ala Lys Ala Ala Asp Lys Ala Ser Val Thr 50 55 60 Gly Ile AlaLys Gly Ile Lys Glu Ile Val Glu Ala Ala Gly Gly Ser 65 70 75 80 Glu LysLeu Lys Val Ala Ala Ala Lys Gly Glu Asn Asn Lys Gly Ala 85 90 95 Gly LysLeu Phe Gly Lys Ala Gly Asp Ala Ala Asn Gly Asp Ser Glu 100 105 110 AlaAla Ser Lys Ala Ala Gly Ala Val Ser Ala Val Ser Gly Glu Gln 115 120 125Ile Leu Ser Ala Ile Val Thr Ala Ala Ala Ala Gly Ala Ala Glu Gln 130 135140 Asp Gly Glu Lys Pro Ala Glu Ala Lys Asn Pro Ile Ala Ala Ala Ile 145150 155 160 Gly Lys Gly Asn Glu Glu Asn Gly Ala Glu Phe Asn Lys Glu GlyMet 165 170 175 Lys Lys Asp Asp Gln Ile Ala Ala Ala Ile Ala Leu Arg GlyMet Ala 180 185 190 Lys Asp Gly Lys Phe Ala Val Lys Lys Asp Asp Glu LysGlu Lys Ala 195 200 205 Glu Gly Ala Ile Lys Gly 210 35 214 PRT Borreliaburgdorferi 35 Ser Thr Thr Gly Lys Pro Asp Ser Thr Gly Ser Val Gly ThrAla Val 1 5 10 15 Glu Gly Ala Ile Lys Glu Val Ser Glu Leu Leu Asp LysLeu Val Lys 20 25 30 Ala Val Lys Thr Ala Glu Gly Ala Ser Ser Gly Thr AspAla Ile Gly 35 40 45 Glu Val Val Ala Asp Asp Asn Ala Ala Lys Val Ala AspLys Ala Ser 50 55 60 Val Thr Gly Ile Ala Lys Gly Ile Lys Glu Ile Val GluAla Ala Gly 65 70 75 80 Gly Ser Glu Lys Leu Lys Val Ala Ala Ala Thr GlyGlu Ser Asn Lys 85 90 95 Gly Ala Gly Lys Leu Phe Gly Lys Val Gly Ala HisAla Gly Asp Ser 100 105 110 Glu Ala Ala Ser Lys Ala Ala Gly Ala Val SerAla Val Ser Gly Glu 115 120 125 Gln Ile Leu Ser Ala Ile Val Lys Ala AlaGly Ala Ala Glu Gln Glu 130 135 140 Gly Lys Lys Pro Glu Glu Ala Lys AsnPro Ile Ala Ala Ala Ile Gly 145 150 155 160 Lys Gly Asp Ala Glu Asn GlyAla Glu Phe Asn His Asp Gly Met Lys 165 170 175 Lys Asp Asp Gln Ile AlaAla Ala Ile Ala Leu Arg Gly Met Ala Lys 180 185 190 Asp Gly Lys Phe AlaVal Lys Ser Gly Gly Gly Glu Lys Gly Lys Ala 195 200 205 Glu Gly Ala IleLys Gly 210 36 215 PRT Borrelia burgdorferi 36 Ser Thr Thr Gly Lys ProAsp Ser Thr Gly Ser Val Gly Thr Ala Val 1 5 10 15 Glu Gly Ala Ile LysGlu Val Ser Glu Leu Leu Asp Lys Leu Val Thr 20 25 30 Ala Val Lys Thr AlaGlu Gly Ala Ser Ser Gly Thr Asp Ala Ile Gly 35 40 45 Glu Val Val Asp AsnAsp Ala Ala Ala Lys Ala Ala Asp Lys Glu Ser 50 55 60 Val Thr Gly Ile AlaLys Gly Ile Lys Glu Ile Val Glu Ala Ala Arg 65 70 75 80 Gly Ser Glu LysLeu Lys Ala Val Ala Ala Ala Lys Glu Gly Asn Glu 85 90 95 Lys Ala Gly LysLeu Phe Gly Lys Ala Gly Ala Ala Ala Gly Asp Ser 100 105 110 Glu Ala AlaSer Lys Ala Ala Gly Ala Val Ser Ala Val Ser Gly Glu 115 120 125 Gln IleLeu Ser Ala Ile Val Lys Ala Ala Ala Ala Ala Ala Gly Asp 130 135 140 GlnGlu Gly Lys Lys Pro Gly Asp Ala Lys Asn Pro Ile Ala Ala Ala 145 150 155160 Ile Gly Lys Gly Asp Ala Asp Asp Gly Ala Glu Phe Asp His Glu Met 165170 175 Lys Lys Asp Asp Gln Ile Ala Ala Ala Ile Ala Leu Arg Gly Met Ala180 185 190 Lys Asp Gly Lys Phe Ala Val Lys Ser Gly Gly Gly Glu Lys GluLys 195 200 205 Ala Glu Gly Ala Ile Lys Gly 210 215 37 211 PRT Borreliaburgdorferi 37 Ser Thr Thr Gly Lys Pro Asp Ser Thr Gly Ser Val Gly ThrAla Val 1 5 10 15 Glu Gly Ala Ile Lys Glu Val Ser Gly Ala Ala Asp LysLeu Val Lys 20 25 30 Ala Val Lys Thr Ala Glu Gly Ala Ser Ser Gly Thr AlaAla Ile Gly 35 40 45 Glu Val Val Asp Asn Asn Asn Ala Ala Lys Ala Ala AspLys Ala Ser 50 55 60 Val Thr Gly Ile Ala Lys Gly Ile Lys Glu Ile Val GluAla Ala Gly 65 70 75 80 Gly Ser Glu Lys Leu Lys Val Ala Ala Ala Thr GlyGlu Ser Asn Lys 85 90 95 Gly Ala Gly Lys Leu Phe Gly Lys Ala Gly Ala AspAla His Gly Asp 100 105 110 Ser Glu Ala Ala Ser Lys Ala Ala Gly Ala ValSer Ala Val Ser Gly 115 120 125 Glu Gln Ile Leu Ser Ala Ile Val Lys AlaAla Asp Ala Ala Asp Gln 130 135 140 Asp Gly Glu Lys Pro Gly Asp Ala LysAsn Pro Ile Ala Ala Ala Ile 145 150 155 160 Gly Lys Gly Asp Gly Gly AlaGlu Phe Ser Gln Asp Glu Met Lys Lys 165 170 175 Asp Asp Gln Ile Ala AlaAla Ile Ala Leu Arg Gly Met Ala Lys Asp 180 185 190 Gly Lys Phe Ala ValLys Asn Asn Glu Lys Gly Lys Ala Glu Gly Ala 195 200 205 Ile Lys Gly 21038 216 PRT Borrelia burgdorferi 38 Ser Thr Thr Gly Lys Pro Asp Ser ThrGly Ser Val Gly Thr Ala Val 1 5 10 15 Glu Gly Ala Ile Lys Glu Val SerGlu Leu Leu Asp Lys Leu Val Lys 20 25 30 Ala Val Lys Thr Ala Glu Glu AlaSer Ser Gly Thr Asp Ala Ile Gly 35 40 45 Glu Val Val Asp Asn Ala Ala AlaAla Lys Ala Ala Asp Lys Ala Ser 50 55 60 Val Thr Gly Ile Ala Lys Gly IleLys Glu Ile Val Glu Ala Ala Gly 65 70 75 80 Gly Ser Glu Lys Leu Lys AlaAla Ala Ala Ala Thr Gly Glu Asn Asn 85 90 95 Lys Glu Ala Gly Lys Leu PheGly Lys Val Asp Ala Gly Asn Ala Gly 100 105 110 Asp Ser Glu Ala Ala SerLys Ala Ala Gly Ala Val Ser Ala Val Ser 115 120 125 Gly Glu Gln Ile LeuSer Ala Ile Val Lys Ala Ala Gly Glu Ala Ala 130 135 140 Gly Asp Gln GluGly Lys Lys Pro Gly Glu Ala Lys Asn Pro Ile Ala 145 150 155 160 Ala AlaIle Gly Lys Gly Asn Ala Asp Asp Gly Ala Glu Phe Gly Asp 165 170 175 GlyMet Lys Lys Asp Asp Gln Ile Ala Ala Ala Ile Ala Leu Arg Gly 180 185 190Met Ala Lys Asp Gly Lys Phe Ala Val Lys Ser Gly Gly Glu Lys Glu 195 200205 Lys Ala Glu Gly Ala Ile Lys Gly 210 215 39 213 PRT Borreliaburgdorferi 39 Ser Thr Thr Gly Lys Pro Asp Ser Thr Gly Ser Val Gly ThrAla Val 1 5 10 15 Glu Gly Ala Ile Lys Glu Val Ser Glu Leu Leu Asp LysLeu Val Lys 20 25 30 Ala Val Lys Thr Ala Glu Gly Ala Ser Ser Gly Thr AspAla Ile Gly 35 40 45 Glu Val Val Ala Asn Ala Gly Ala Ala Lys Val Ala AspLys Ala Ser 50 55 60 Val Thr Gly Ile Ala Lys Gly Ile Lys Glu Ile Val GluAla Ala Gly 65 70 75 80 Gly Ser Glu Lys Leu Lys Val Ala Ala Ala Thr GlyGlu Ser Asn Lys 85 90 95 Gly Ala Gly Lys Leu Phe Gly Lys Val Asp Asp AlaHis Ala Gly Asp 100 105 110 Ser Glu Ala Ala Ser Lys Ala Ala Gly Ala ValSer Ala Val Ser Gly 115 120 125 Glu Gln Ile Leu Ser Ala Ile Val Lys AlaAla Asp Ala Ala Glu Gln 130 135 140 Asp Gly Lys Lys Pro Glu Glu Ala ThrAsn Pro Ile Ala Ala Ala Ile 145 150 155 160 Gly Glu Gly Asn Glu Asp GlyAla Asp Phe Gly Lys Asp Glu Met Lys 165 170 175 Lys Asp Asp Gln Ile AlaAla Ala Ile Ala Leu Arg Gly Met Ala Lys 180 185 190 Asp Gly Lys Phe AlaVal Lys Asp Gly Gly Glu Lys Gly Lys Ala Glu 195 200 205 Gly Ala Ile LysGly 210 40 211 PRT Borrelia burgdorferi 40 Ser Thr Thr Gly Lys Pro AspSer Thr Gly Ser Val Gly Thr Ala Val 1 5 10 15 Glu Gly Ala Ile Lys GluVal Ser Glu Leu Leu Asp Lys Leu Val Lys 20 25 30 Ala Val Lys Thr Ala GluGly Ala Ser Ser Gly Thr Ala Ala Ile Gly 35 40 45 Glu Val Val Asp Asn AspAla Lys Ala Ala Asp Lys Ala Ser Val Thr 50 55 60 Gly Ile Ala Lys Gly IleLys Glu Ile Val Glu Ala Ala Gly Gly Ser 65 70 75 80 Glu Lys Leu Lys AlaVal Ala Ala Ala Lys Gly Glu Asn Asn Lys Gly 85 90 95 Ala Gly Lys Leu PheGly Lys Val Asp Asp Ala His Ala Gly Asp Ser 100 105 110 Glu Ala Ala SerLys Ala Ala Gly Ala Val Ser Ala Val Ser Gly Glu 115 120 125 Gln Ile LeuSer Ala Ile Val Thr Ala Ala Asp Ala Ala Glu Gln Asp 130 135 140 Gly GluLys Pro Glu Glu Ala Lys Asn Pro Ile Ala Ala Ala Ile Gly 145 150 155 160Lys Gly Asn Glu Asp Gly Ala Glu Phe Gly Lys Asp Glu Met Lys Lys 165 170175 Asp Asp Gln Ile Ala Ala Ala Ile Ala Leu Arg Gly Met Ala Lys Asp 180185 190 Gly Lys Phe Ala Val Lys Asp Gly Glu Lys Glu Lys Ala Glu Gly Ala195 200 205 Ile Lys Gly 210 41 212 PRT Borrelia burgdorferi 41 Ser ThrThr Gly Lys Pro Asp Ser Thr Gly Ser Val Gly Thr Ala Val 1 5 10 15 GluGly Ala Ile Lys Glu Val Ser Glu Leu Leu Asp Lys Leu Val Lys 20 25 30 AlaVal Lys Thr Ala Glu Gly Ala Ser Ser Gly Thr Ala Ala Ile Gly 35 40 45 GluVal Val Ala Asp Asp Asn Ala Ala Lys Val Ala Asp Lys Asp Ser 50 55 60 ValLys Gly Ile Ala Lys Gly Ile Lys Glu Ile Val Glu Ala Ala Gly 65 70 75 80Gly Ser Lys Lys Leu Lys Val Ala Ala Ala Lys Glu Gly Asn Glu Lys 85 90 95Ala Gly Lys Leu Phe Gly Lys Val Asp Asp Ala Ala His Ala Gly Asp 100 105110 Ser Glu Ala Ala Ser Lys Ala Ala Gly Ala Val Ser Ala Val Ser Gly 115120 125 Glu Gln Ile Leu Ser Ala Ile Val Thr Ala Ala Asp Ala Ala Glu Gln130 135 140 Asp Gly Glu Lys Pro Gly Asp Ala Lys Asn Pro Ile Ala Ala AlaIle 145 150 155 160 Gly Lys Gly Asn Ala Asp Gly Ala Glu Phe Lys Asp GluMet Lys Lys 165 170 175 Asp Asp Gln Ile Ala Ala Ala Ile Ala Leu Arg GlyMet Ala Lys Asp 180 185 190 Gly Lys Phe Ala Val Lys Asn Asp Asp Glu LysGly Lys Ala Glu Gly 195 200 205 Ala Ile Lys Gly 210 42 209 PRT Borreliaburgdorferi 42 Ser Thr Thr Gly Lys Pro Asp Ser Thr Gly Ser Val Gly ThrAla Val 1 5 10 15 Glu Gly Ala Ile Lys Glu Val Ser Glu Leu Leu Asp LysLeu Val Lys 20 25 30 Ala Val Lys Thr Ala Glu Gly Ala Ser Ser Gly Thr AspAla Ile Gly 35 40 45 Glu Val Val Asp Asn Ala Ala Lys Ala Ala Asp Lys AlaSer Val Thr 50 55 60 Gly Ile Ala Lys Gly Ile Lys Glu Ile Val Glu Ala AlaGly Gly Ser 65 70 75 80 Glu Lys Leu Lys Ala Val Ala Ala Ala Thr Gly GluAsn Asn Lys Gly 85 90 95 Ala Gly Lys Leu Phe Gly Lys Ala Gly Ala Gly AlaGly Gly Asp Ser 100 105 110 Glu Ala Ala Ser Lys Ala Ala Gly Ala Val SerAla Val Ser Gly Glu 115 120 125 Gln Ile Leu Ser Ala Ile Val Thr Ala AlaAsp Ala Ala Glu Gln Asp 130 135 140 Gly Lys Lys Pro Glu Glu Ala Lys AsnPro Ile Ala Ala Ala Ile Gly 145 150 155 160 Asn Lys Asp Gly Gly Ala AspPhe Gly Asp Gly Met Lys Lys Asp Asp 165 170 175 Gln Ile Ala Ala Ala IleAla Leu Arg Gly Met Ala Lys Asp Gly Lys 180 185 190 Phe Ala Val Lys GluAsp Glu Lys Glu Lys Ala Glu Gly Ala Ile Lys 195 200 205 Gly 43 212 PRTBorrelia burgdorferi 43 Ser Thr Thr Gly Lys Pro Asp Ser Thr Gly Ser ValGly Thr Ala Val 1 5 10 15 Glu Gly Ala Ile Lys Glu Val Ser Glu Leu LeuAsp Lys Leu Val Lys 20 25 30 Ala Val Lys Thr Ala Glu Gly Ala Ser Ser GlyThr Ala Ala Ile Gly 35 40 45 Glu Val Val Ala Asp Ala Asp Ala Ala Lys ValAla Asp Lys Ala Ser 50 55 60 Val Lys Gly Ile Ala Lys Gly Ile Lys Glu IleVal Glu Ala Ala Gly 65 70 75 80 Gly Ser Glu Lys Leu Lys Ala Val Ala AlaAla Lys Gly Glu Asn Asn 85 90 95 Lys Gly Ala Gly Lys Leu Phe Gly Lys AlaGly Ala Ala Ala His Gly 100 105 110 Asp Ser Glu Ala Ala Ser Lys Ala AlaGly Ala Val Ser Ala Val Ser 115 120 125 Gly Glu Gln Ile Leu Ser Ala IleVal Thr Ala Ala Asp Ala Ala Glu 130 135 140 Gln Asp Gly Lys Lys Pro GluGlu Ala Lys Asn Pro Ile Ala Ala Ala 145 150 155 160 Ile Gly Asp Lys AspGly Gly Ala Glu Phe Gly Gln Asp Glu Met Lys 165 170 175 Lys Asp Asp GlnIle Ala Ala Ala Ile Ala Leu Arg Gly Met Ala Lys 180 185 190 Asp Gly LysPhe Ala Val Lys Asp Gly Glu Lys Glu Lys Ala Glu Gly 195 200 205 Ala IleLys Gly 210 44 212 PRT Borrelia burgdorferi 44 Ser Thr Thr Gly Lys ProAsp Ser Thr Gly Ser Val Gly Thr Ala Val 1 5 10 15 Glu Gly Ala Ile LysGlu Val Ser Glu Leu Leu Asp Lys Leu Val Lys 20 25 30 Ala Val Lys Thr AlaGlu Gly Ala Ser Ser Gly Thr Asp Ala Ile Gly 35 40 45 Glu Val Val Ala AsnAla Gly Ala Ala Lys Val Ala Asp Lys Ala Ser 50 55 60 Val Thr Gly Ile AlaLys Gly Ile Lys Glu Ile Val Glu Ala Ala Gly 65 70 75 80 Gly Ser Glu LysLeu Lys Ala Val Ala Ala Lys Thr Glu Asn Ser Lys 85 90 95 Gly Ala Gly LysLeu Phe Gly Lys Val Asp Asp Ala His Ala Gly Asp 100 105 110 Ser Glu AlaAla Ser Lys Ala Ala Gly Ala Val Ser Ala Val Ser Gly 115 120 125 Glu GlnIle Leu Ser Ala Ile Val Thr Ala Ala Ala Ala Gly Glu Gln 130 135 140 AspGly Glu Lys Pro Gly Asp Ala Lys Asn Pro Ile Ala Ala Ala Ile 145 150 155160 Gly Lys Gly Asp Ala Asp Asp Gly Ala Asp Phe Gly Asp Gly Met Lys 165170 175 Lys Asp Asp Gln Ile Ala Ala Ala Ile Ala Leu Arg Gly Met Ala Lys180 185 190 Asp Gly Lys Phe Ala Val Lys Asn Asp Glu Lys Gly Lys Ala GluGly 195 200 205 Ala Ile Lys Gly 210 45 209 PRT Borrelia burgdorferi 45Ser Thr Thr Gly Lys Pro Asp Ser Thr Gly Ser Val Gly Thr Ala Val 1 5 1015 Glu Gly Ala Ile Lys Glu Val Ser Glu Leu Leu Asp Lys Leu Val Lys 20 2530 Ala Val Lys Thr Ala Glu Gly Ala Ser Ser Gly Thr Asp Ala Ile Gly 35 4045 Glu Val Val Asp Asn Asp Ala Lys Val Ala Asp Lys Ala Ser Val Thr 50 5560 Gly Ile Ala Lys Gly Ile Lys Glu Ile Val Glu Ala Ala Gly Gly Ser 65 7075 80 Glu Lys Leu Lys Ala Val Ala Ala Lys Thr Glu Asn Asn Lys Gly Glu 8590 95 Gly Lys Leu Phe Gly Lys Ala Gly Ala Asp Ala Asn Gly Asp Ser Glu100 105 110 Ala Ala Ser Lys Ala Ala Gly Ala Val Ser Ala Val Ser Gly GluGln 115 120 125 Ile Leu Ser Ala Ile Val Lys Ala Ala Gly Ala Ala Glu GlnAsp Gly 130 135 140 Glu Lys Pro Gly Asp Ala Thr Asn Pro Ile Ala Ala AlaIle Gly Asp 145 150 155 160 Lys Asp Gly Asp Ala Glu Phe Gly Asp Gly MetLys Lys Asp Asp Gln 165 170 175 Ile Ala Ala Ala Ile Ala Leu Arg Gly MetAla Lys Asp Gly Lys Phe 180 185 190 Ala Val Lys Gly Asn Asn Glu Lys GlyLys Ala Glu Gly Ala Ile Lys 195 200 205 Gly 46 216 PRT Borreliaburgdorferi 46 Ser Thr Thr Gly Lys Pro Asp Ser Thr Gly Ser Val Gly ThrAla Val 1 5 10 15 Glu Gly Ala Ile Lys Glu Val Ser Glu Leu Leu Asp LysLeu Val Lys 20 25 30 Ala Val Lys Thr Ala Glu Gly Ala Ser Ser Gly Thr AlaAla Ile Gly 35 40 45 Glu Val Val Ala Asp Ala Ala Lys Ala Ala Asp Lys AspSer Val Lys 50 55 60 Gly Ile Ala Lys Gly Ile Lys Glu Ile Val Glu Ala AlaGly Gly Ser 65 70 75 80 Glu Lys Leu Lys Ala Val Ala Ala Ala Lys Thr GluAsn Ser Lys Gly 85 90 95 Ala Gly Lys Leu Phe Gly Lys Val Asp Gly Ala AlaHis Gly Asp Ser 100 105 110 Glu Ala Ala Ser Lys Ala Ala Gly Ala Val SerAla Val Ser Gly Glu 115 120 125 Gln Ile Leu Ser Ala Ile Val Thr Ala AlaGly Ala Ala Ala Ser Glu 130 135 140 Ala Asp Gln Glu Gly Lys Lys Pro AlaAsp Ala Thr Asn Pro Ile Ala 145 150 155 160 Ala Ala Ile Gly Lys Gly AsnGlu Glu Asn Gly Ala Glu Phe Gly Asp 165 170 175 Gly Met Lys Lys Asp AspGln Ile Ala Ala Ala Ile Ala Leu Arg Gly 180 185 190 Met Ala Lys Asp GlyLys Phe Ala Val Lys Asn Asp Asp Glu Lys Gly 195 200 205 Lys Ala Glu GlyAla Ile Lys Gly 210 215 47 208 PRT Borrelia burgdorferi 47 Ser Thr ThrGly Lys Pro Asp Ser Thr Gly Ser Val Gly Thr Ala Val 1 5 10 15 Glu GlyAla Ile Lys Glu Val Ser Glu Leu Leu Asp Lys Leu Val Thr 20 25 30 Ala ValLys Thr Ala Glu Gly Ala Ser Ser Gly Thr Asp Ala Ile Gly 35 40 45 Glu ValVal Asp Asn Asp Ala Lys Ala Ala Asp Lys Ala Ser Val Thr 50 55 60 Gly IleAla Lys Gly Ile Lys Glu Ile Val Glu Ala Ala Gly Gly Ser 65 70 75 80 GluLys Leu Lys Ala Ala Ala Ala Lys Gly Glu Asn Ser Lys Gly Ala 85 90 95 GlyLys Leu Phe Gly Lys Ala Gly Ala Asp Ala Asn Gly Asp Ser Glu 100 105 110Ala Ala Ser Lys Ala Ala Gly Ala Val Ser Ala Val Ser Gly Glu Gln 115 120125 Ile Leu Ser Ala Ile Val Thr Ala Ala Asp Ala Ala Glu Gln Asp Gly 130135 140 Glu Lys Pro Gly Asp Ala Lys Asn Pro Ile Ala Ala Ala Ile Gly Lys145 150 155 160 Gly Asp Gly Asp Ala Asp Phe Gly Asp Gly Met Lys Lys AspAsp Gln 165 170 175 Ile Ala Ala Ala Ile Ala Leu Arg Gly Met Ala Lys AspGly Lys Phe 180 185 190 Ala Val Lys Asn Asp Glu Lys Gly Lys Ala Glu GlyAla Ile Lys Gly 195 200 205 48 212 PRT Borrelia burgdorferi 48 Ser ThrThr Gly Lys Pro Asp Ser Thr Gly Ser Val Gly Thr Ala Val 1 5 10 15 GluGly Ala Ile Lys Glu Val Ser Glu Leu Leu Asp Lys Leu Val Lys 20 25 30 AlaVal Lys Thr Ala Glu Gly Ala Ser Ser Gly Thr Ala Ala Ile Gly 35 40 45 GluVal Val Asp Asn Ala Ala Lys Ala Ala Asp Lys Ala Ser Val Thr 50 55 60 GlyIle Ala Lys Gly Ile Lys Glu Ile Val Glu Ala Ala Gly Gly Ser 65 70 75 80Glu Lys Leu Lys Ala Val Ala Ala Ala Lys Thr Glu Asn Ser Lys Gly 85 90 95Ala Gly Lys Leu Phe Gly Lys Ala Gly Ala Asp Ala Asn Ala Gly Asp 100 105110 Ser Glu Ala Ala Ser Lys Ala Ala Gly Ala Val Ser Ala Val Ser Gly 115120 125 Glu Gln Ile Leu Ser Ala Ile Val Thr Ala Ala Asp Ala Ala Glu Gln130 135 140 Glu Gly Lys Lys Pro Glu Glu Ala Lys Asn Pro Ile Ala Ala AlaIle 145 150 155 160 Gly Lys Gly Asn Ala Asp Asp Gly Ala Asp Phe Gly GlnAsp Met Lys 165 170 175 Lys Asp Asp Gln Ile Ala Ala Ala Ile Ala Leu ArgGly Met Ala Lys 180 185 190 Asp Gly Lys Phe Ala Val Lys Lys Asp Glu LysGly Lys Ala Glu Gly 195 200 205 Ala Ile Lys Gly 210

What is claimed is:
 1. An isolated and purified nucleic acid segmentthat encodes a polypeptide comprising an amino acid residue sequence ofSEQ ID NO:2, or its complement.
 2. The nucleic acid segment of claim 1,wherein said nucleic acid segment is identical or complementary to anucleic acid segment of at least 15 contiguous nucleotides of SEQ IDNO:1.
 3. The nucleic acid segment of claim 1, wherein said nucleic acidsegment hybridizes to the sequence of SEQ ID NO:1, or its complement,under conditions of high stringency.
 4. The nucleic acid segment ofclaim 1, wherein said nucleic acid segment hybridizes to a nucleic acidsegment that encodes SEQ ID NO:2, or to the complement of such asequence.
 5. The nucleic acid segment of claim 1, wherein said nucleicacid segment further encodes additional amino acids at the N or Cterminal end of the amino acid residue sequence.
 6. The nucleic acidsegment of claim 1, wherein said nucleic acid segment further comprisesadditional terminal 5′ or 3′ nucleotide sequences or internal nucleotidesequences.
 7. An isolated nucleic acid segment comprising at least 15contiguous bases of SEQ ED NO:1, or the complement thereof.
 8. Thenucleic acid segment of claim 7, wherein the nucleic acid segmentcomprises at least 20 contiguous nucleotides from SEQ ID NO:1, or thecomplement thereof.
 9. The nucleic acid segment of claim 8, wherein thenucleic acid segment comprises at least 30 contiguous nucleotides fromSEQ ID NO:1, or the complement thereof.
 10. The nucleic acid segment ofclaim 9, wherein the nucleic acid segment comprises at least 40contiguous nucleotides from SEQ ID NO:1 or the complement thereof. 11.The nucleic acid segment of claim 10, wherein the nucleic acid segmentcomprises at least 50 contiguous nucleotides from SEQ ID NO:1, or thecomplement thereof.
 12. The nucleic acid segment of claim 11, whereinthe nucleic acid segment comprises at least 75 contiguous nucleotidesfrom SEQ ID NO:1, or the complement thereof.
 13. The nucleic acidsegment of claim 12, wherein the nucleic acid segment comprises at least100 contiguous nucleotides from SEQ ID NO1, or the complement thereof.14. The nucleic acid segment of claim 13, wherein the nucleic acidsegment comprises at least 200 contiguous nucleotides from SEQ ID NO:1,or the complement thereof.
 15. The nucleic acid segment of claim 14,wherein the nucleic acid segment comprises at least 500 contiguousnucleotides from SEQ ID NO:1, or the complement thereof.
 16. The nucleicacid segment of claim 15, wherein the nucleic acid segment comprises atleast 1000 contiguous nucleotides from SEQ ID NO:1, or the complementthereof.
 17. The nucleic acid segment of claim 16, wherein the nucleicacid segment comprises at least 1227 contiguous nucleotides from SEQ IDNO:1, or the complement thereof.
 18. The nucleic acid segment of claim7, wherein said nucleic acid segment encodes a polypeptide of at least50 amino acids in length.
 19. The nucleic acid segment of claim 18,wherein said nucleic acid segment encodes a polypeptide of at least 100amino acids in length.
 20. The nucleic acid segment of claim 19, whereinsaid nucleic acid segment encodes a polypeptide of at least 150 aminoacids in length.
 21. The nucleic acid segment of claim 20, wherein saidnucleic acid segment encodes a polypeptide of at least 200 amino acidsin length.
 22. The nucleic acid segment of claim 21, wherein saidnucleic acid segment encodes a polypeptide of at least 400 amino acidsin length.
 23. An isolated nucleic acid segment comprising at least 15contiguous nucleotides from SEQ ID NO:1 or its complement, wherein saidnucleic acid segment is further defined as at least 100 bases in length.24. The nucleic acid segment of claim 23, wherein said nucleic acidsegment is at least 500 bases in length.
 25. The nucleic acid segment ofclaim 24, wherein said nucleic acid segment is at least 1,000 bases inlength.
 26. The nucleic acid segment of claim 25, wherein said nucleicacid segment is at least 2,000 bases in length.
 27. The nucleic acidsegment of claim 26, wherein said nucleic acid segment is at least 3,000bases in length.
 28. The nucleic acid segment of claim 27, wherein saidnucleic acid segment is at least 5,000 bases in length.
 29. The nucleicacid segment of claim 28, wherein said nucleic acid segment is at least10,000 bases in length.
 30. A recombinant host cell comprising a nucleicacid segment comprising at least 20 contiguous bases of SEQ ID NO:1, orthe complement thereof.
 31. The recombinant host cell of claim 30,wherein said nucleic acid segment comprises at least 30 contiguous basesof SEQ ID NO:1, or the complement thereof.
 32. The recombinant host cellof claim 31, wherein said nucleic acid segment comprises at least 40contiguous bases of SEQ ID NO:1, or the complement thereof.
 33. Therecombinant host cell of claim 32, wherein said nucleic acid segmentcomprises at least 1227 contiguous bases of SEQ ID NO:1, or thecomplement thereof.
 34. The recombinant host cell of claim 33, whereinsaid nucleic acid segment encodes the amino acid sequence of SEQ IDNO:2, or its complement.
 35. An expression vector comprising a nucleicacid segment that encodes a polynucleotide comprising at least 15contiguous bases of SEQ ID NO:
 1. 36. The expression vector of claim 35,wherein said nucleic acid segment is operatively linked to a promotor.37. The expression vector of claim 35, wherein said nucleic acid segmentencodes the amino acid residue sequence of SEQ ID NO:2.
 38. An isolatedand purified nucleic acid segment comprising at least nucleotides 75 to1142 of SEQ ID NO:1, or the complement thereof.
 39. The nucleic acidsegment of claim 38, wherein said nucleic acid segment comprising atleast nucleotides 75 to 1142 of SEQ ID NO:1, or its complement, isoperatively linked to a promoter.
 40. A recombinant host cell comprisinga nucleic acid segment comprising at least nucleotides 75 to 1142 of SEQID NO:1, or the complement thereof.
 41. The recombinant host cell ofclaim 40, wherein said nucleic acid segment comprising at leastnucleotides 75 to 1142 of SEQ ID NO:1, or its complement, is operativelylinked to a promoter.
 42. An expression vector comprising a nucleic acidsegment comprising at least nucleotides 75 to 1142 of SEQ ID NO:1, orthe complement thereof.
 43. The expression vector of claim 42, whereinsaid nucleic acid segment comprising at least nucleotides 75 to 1142 ofSEQ ID NO:1, or its complement, is operatively linked to a promoter. 44.A method for diagnosing Lyme disease in a subject, comprising assayingfor a nucleic acid segment having a nucleotide sequence comprising atleast 15 contiguous bases of SEQ ID NO:1, or its complement.
 45. Themethod of claim 44, wherein said subject is a human.
 46. The method ofclaim 44, wherein said subject is a dog, a deer or a mouse.
 47. Themethod of claim 44, wherein said nucleic acid segment is identified byhybridization.
 48. The method of claim 44, wherein said nucleic acidsegment is identified by PCR.